tetrapyrrole biosynthesis 
 is one of the most highly conserved metabolic 
 pathways in nature , and its final products such as heme , chlorophyll , 
 or corrins fulfill vital functions in nearly all living organisms . 

a blockade of this pathway is correlated with detrimental effects 
 not only in man , as documented by various genetic porphyric disorders 
 and lead poisoning , but also in many human pathogenic 
 infections .
eukaryotic organisms unable to synthesize heme , such 
 as several unicellular parasites or multicellular nematodes , have 
 molecular transporters to sequester heme from their environment or 
 host . for non - heme
auxotrophic organisms , heme 
 biosynthesis represents a suitable target for antiparasitic or antibacterial 
 drugs with the precondition that the drug candidate only interferes 
 with tetrapyrrole biosynthesis in the pathogen and not in the host .
one heme biosynthesis enzyme that shows a profound divergence in 
 its molecular properties between different species is porphobilinogen 
 synthase ( e.c .
pbgs synthesizes 
 porphobilinogen by the asymmetric condensation of two molecules of 
 5-aminolevulinic acid ( 5-ala ) , which is the first common step of tetrapyrrole 
 biosynthesis . despite high sequence conservation , 

pbgs orthologs differ dramatically in their metal cofactor requirements as well as in the stability of different quaternary 
 structures .
pbgs is a homooligomeric protein 
 with single subunits adopting an ( /)8-barrel 
 fold and an extended n - terminal arm that is essential for subunit  subunit 
 interactions . under varying environmental conditions ,
the subunits 
 can adopt different conformations that support assembly into different 
 quaternary structures with distinct catalytic activities ; i.e. , pbgs 
 is a morpheein .
mammalian , yeast , and many bacterial 
 enzymes have a cys - rich sequence motif that complexes catalytically 
 essential zn ( in the literature often referred to as 
 metalb or znb site ; see also sequence alignment 
 in figure s1 in supporting information ) 
 required for binding of the second 5-ala substrate molecule . in the 
 plant ( chloroplast ) and other bacterial enzymes , this motif is replaced 
 by a glu - rich sequence rendering enzymatic activity of these proteins 
 zn - independent . for some zn - independent 
 proteins ( bradyrhizobium japanicum , pisum sativum , toxoplasma gondii ) 
 a requirement for tightly binding catalytic mg at the 
 active site has been suggested , while other proteins are catalytically 
 active in the absence of divalent metal ions ( pseudomonas 
 aeruginosa , plasmodium 
 falciparum , rhodobacter 
 capsulatus ) .
furthermore many 
 orthologs are allosterically activated by mg binding 
 to a conserved glu residue ( e245 of p. aeruginosa pbgs ( papbgs ) ) at 
 an octamer - specific arm - to - barrel interface between adjacent subunits . 

this includes almost all zn - independent and many zn - dependent orthologs but not the human pbgs ( hspbgs ) or that of any metazoa or fungi .
binding of mg to the allosteric mgc site 
 stabilizes an octameric quaternary structure with high activity which 
 is in equilibrium with dimeric or hexameric assemblies that have low 
 activity .
the octameric assembly contains 
 subunits in a conformation that allows intersubunit interactions necessary 
 for closure of the active site lid , enabling catalysis as observed 
 for papbgs .
we have recently reported that trisubstituted benzimidazoles 
 of 
 the waladin1 family are a new class of species - discriminatory pbgs 
 inhibitors .
waladin1 is a specific inhibitor 
 of the mg - responsive pbgs of the -proteobacterium wolbachia , endosymbiont in the filarial nematode parasite brugia malayi , which is a drug target for treating filarial 
 infections .
waladin1 is a mixed competitive / noncompetitive inhibitor that interferes 
 with the induction of enzymatic activity by mg .
given the specificity of this inhibitory mode 
 of action and the insensitivity of hspbgs to this 
 compound , waladin1 may also be a specific inhibitor of other pbgs 
 orthologs that are responsive to mg ( depending on either 
 a catalytic or allosteric mg ) .
hence , we have characterized 
 the activity of the benzimidazole waladin1 , several waladin1 derivatives , 
 and the tricyclic quinoline derivative waladin2 in a number of mg - stimulated pbgs orthologs . 

these included the human pathogenic or opportunistic bacteria escherichia coli ( ec ) , vibrio cholerae ( vc ) , yersinia enterocolitica ( ye ) , p. aeruginosa ( pa ) , the apicomplexan parasite toxoplasma gondii ( tg ) , and the chloroplast protein of pisum sativum ( ps ) . as a further mg - independent 
 ortholog , pbgs from the fruit fly drosophila melanogaster ( dm ) was analyzed .
depending on the pbgs class , 
 waladins could inhibit or stimulate activity or have no effect .
incubation 
 of waladin1 with the p. aeruginosa enzyme resulted 
 in an inhibitory or stimulatory effect depending on the experimental 
 conditions .
our findings suggest that modulation of pbgs activity 
 by waladins is likely an allosteric process that may drive the oligomeric 
 equilibrium of these structurally flexible proteins toward a more 
 active or less active assembly .
the inhibitory profile of waladin1 ( 1 ) , derivatives thereof ( 214 ) , and waladin2 ( 15 ) ( figure 1a  c , table 1 ) against different pbgs orthologs was characterized using standardized 
 assay conditions for each protein with constant concentrations of 
 1 mm mgcl2 ( except dmpbgs ) and 200 m 
 5-ala . on the basis of the outcome of modulation by waladins , pbgs 
 enzymes
may be assigned to different groups ( figure 2 ) : group x orthologs ( containing the plant pspbgs and -protobacterial wpbgs ) were inhibited 
 by waladins ( incl .
waladin2 ) , while group y orthologs ( containing 
 the -proteobacterial and tgpbgs ) were stimulated . 
 finally , group z orthologs , containing zn - dependent pbgs 
 orthologs from d. melanogaster and h. sapiens , were insensitive to waladin1 ( ic50 > 500 m ) .
chemical 
 structures of waladin inhibitors : ( a ) chemical structure 
 of waladin1 ; ( b ) scaffold of waladin1 benzimidazoles described in 
 table 1 with atom numbering ; ( c ) chemical structure 
 of waladin2 .
three classes of pbgs 
 ortholog groups according to the structure  activity 
 relationship of waladin1 .
group x pbgs orthologs from wolbachia and p. sativum are inhibited by waladin1 benzimidazoles . 

group y pbgs orthologs from e. coli , v. cholera , y. enterocolitica , p. aeruginosa , and t. gondii are stimulated by waladin1 benzimidazoles . 

the metazoan group z pbgs orthologs from d. melanogaster and h. sapiens are insensitive to waladin1 benzimidazoles . 

the asterisk (  ) indicates that papbgs 
 was originally assigned to group y , but it must also , in part , be 
 counted among group x because it was inhibited by waladin1 under certain 
 experimental conditions ( see figure 4 , figure s4 ) , and it is therefore also listed in 
 gray below the inhibited group x orthologs .
no inhibitory activity ; 
 ic50  500 m for pspbgs , wpbgs , hspbgs ; ic50  
 1 mm for dmpbgs , wpbgs , and hspbgs . 
 for dmpbgs ic  1 mm .
waladin1 inhibited the plant p. sativum pbgs ( pspbgs ) with an ic50 of 10.6  
 0.7 m and was equivalent to the ic50 of 11.1 m 
 reported for the wolbachia enzyme ( wpbgs ) .
as the overall inhibitory profile 
 of the benzimidazole derivatives against pspbgs was 
 very similar to that of wpbgs , with some minor differences 
 ( summarized in table 1 and figure 3a ) , these two orthologs were classified as group 
 x. removal of the 2-[(2-thienylcarbonyl)amino]ethyl ) substituent 
 at r ( 3 ) did not significantly alter inhibitory 
 activity against either ortholog with an ic50 of 18.5  
 2.4 for pspbgs and 13.0 m for wpbgs . moving the r - cooh from the c5 to c4 position of 

the benzimidazole led to a complete loss of inhibitory activity ; a 
 positional change to c6 ( 4 ) involved an 8-fold 
 reduction of inhibitory potency ( 28-fold for wpbgs ) .
attachment of the carboxyl function to c7 ( 6 ) 
 also abrogated inhibitory activity for pspbgs , which 
 had not been observed for wpbgs .
alterations in the 
 3-cf3-benzyl r substituent led to a quantitatively 
 similar impairment of inhibitory activity in both orthologs .
however , 
 compound 13 with a methyl group as the r substituent 
 had weak inhibitory activity against pspbgs ( ic50 = 257  46 m ) but not against wpbgs .
waladin2 ( 15 ) was 10-fold less potent 
 at inhibiting pspbgs ( ic50 = 88.2  
 8.0 m ) compared with wpbgs ( ic50 = 8.1 m  ) . summary of 
 sar on pbgs groups x , y , and z : ( a ) sar data for inhibition 
 of group x ( wolbachia and p. sativum ) ; ( b ) stimulation of group y orthologs ( e.coli , p. aeruginosa , v. cholerae , y. 
 enterocolitica , and t. gondii ) ; ( c ) insensitive 
 group z containing hspbgs and dmpbgs . the benzimidazole core is shown in black , and substituents 
 of the parent molecule waladin1 are in blue .
positional changes of 
 residues are indicated by dashed arrows , and chemical changes in substituents 
 are indicated with black solid arrows .
the consequences of chemical 
 alterations with respect to biological activity are highlighted in 
 color : green ( similar or improved activity ) , orange ( reduced activity ) , 
 or purple ( abrogated activity ) . when the effects of a structural modification 
 varied for different members of a group , the species abbreviations 
 are given in the corresponding colored font : w = wolbachia , ps = p. sativum , ec = e. coli , vc = v. cholerae , ye = y. 
 enterocolitica , pa = p. aeruginosa , tg = t. gondii , hs = h. sapiens , dm = d. 
 melanogaster . data from wpbgs and hspbgs were reported in ref ( 21 ) .
elucidation of the mode of action of waladin1 against pspbgs determined a mixed competitive / noncompetitive mechanism 
 and 
 partial competition with the activation induced by mg ( parts a  c of figure s2 ) as seen 
 with the wolbachia protein . at a saturating concentration of 10 mm 5-ala
, waladin1 also induced 
 a decrease of the maximum activity of wpbgs in a 
 mg - response curve ( part d of figure 
 s2 ) .
thus , the effect of waladin1 on wpbgs 
 involves both a reduction in affinity for mg and a reduction 
 in enzymatic velocity when mg is bound . at 100 m 
 waladin1
the other pbgs orthologs tested were from the 
 -proteobacteria e.coli , p. aeruginosa , v. cholerae , y. enterocolitica , and the apicomplexan parasite t. gondii .
these 
 orthologs were stimulated rather than inhibited by waladin1 and several 
 other waladin derivatives and therefore were assigned to group y. 
 the compounds that exerted a stimulating effect on these enzymes were 
 waladin1 , the r positional isomers 4 ( r - cooh at c6 , for ye and tg only ) , 5 ( r - cooh at c4 ) , 6 ( r - cooh at c7 , for ye and tg only ) , and the r positional isomer 7 ( r-4-cf3-benzyl ) ( table 2 , 
 figure 3b ) .
enzymatic activity was stimulated 
 to a maximum of 1542% over control reactions treated with 
 6.7% dmso , corresponding to ec50 values between 20 and 
 300 m according to nonlinear regression ( nlr ) analysis .
nlr 
 gave in part weak fits ( r = 0.640.92 ) 
 probably due to the low extent of stimulation in combination with 
 high ec50 values .
inhibitory 
 chemotype  described above for ps / wpbgs , the exchange of the r-2[(2-thienylcarbonyl)amino]ethyl ) 
 moiety to -h was not tolerated .
although derivative 5 ( r - cooh at c4 ) was completely inactive against ps / wpbgs , it was , together with compound 7 ( r-4-cf3-benzyl ) , the most potent 
 stimulator of enzymatic activity .
vcpbgs 
 was weakly inhibited by compounds 12 ( ic50  1400  624 m ; r = 0.6392 ) and 13 ( ic50  843  
 236 m ; r = 0.8639 ) .
activity data were normalized , and 
 control reactions performed in the presence of 6.7% dmso instead of 
 compound were set to 100% activity .
data from the highest concentration 
 tested ( 533 m ) were excluded from nonlinear regression because 
 biological activity started to decline , and thus , the nonlinear fit 
 of the data was ambiguous .
the corresponding 
 value is reported as the maximum stimulation value , although higher 
 concentrations of compound may lead to a further increase in enzymatic 
 activity . because of their low potency , no ec50 values 
 could be determined for these compounds .
the activity 
 of the zn - dependent fruit fly dmpbgs 
 ortholog was insensitive to waladin1 ( ic50 > 1000 m , 
 table 1 ) and is therefore assigned to group 
 z , along with the likewise waladin1-insensitive hspbgs ( as reported in ref ( 21 ) ) .
dmpbgs and hspbgs have 
 a very similar sar profile ( table 1 , figure 3c ) with the majority of waladin1 derivatives featuring 
 the r- 2[(2-thienylcarbonyl)amino]ethyl ) moiety 
 being ineffective .
the only exceptions are 2 ( with an 
 r - h instead of 3-cf3-benzyl ; ic50(dmpbgs ) = 448  45 m ) and 4 ( a positional isomer with r - cooh at c6 ; ic50(dmpbgs ) = 257  21 m ) that 
 inhibit dmpbgs but not hspbgs . as 
 described previously for hspbgs , also for dmpbgs , replacement of the r-2[(2-thienylcarbonyl)amino]ethyl ) by a hydrogen atom in 3 led to a 3-fold gain in inhibitory activity ( ic50 = 340  31 m ) compared to the parent molecule . 
 within the series of r - h compounds with alterations in 
 the r - position permissive for inhibition of hspbgs , only r-3-ch3-benzyl ( 11 , ic50 = 371  45 m ) ,
r-3-o - ch3 ( 12 , ic50 = 370  93 m ) , 
 and weakly r-4-cf3-benzyl ( 8 , 
 ic50 = 620  93 m ) but not r-2-cf3-benzyl ( 9 ) or r - benzyl ( 10 ) were tolerated by dmpbgs .
multiple sequence alignment of the full 
 length proteins did not reveal molecular motifs that could clearly 
 define group membership ( figure s1 ) .
while 
 all orthologs assigned to either group x or group y feature an allosteric 
 mgc , neither of the metazoan group z orthologs responds 
 to mg ; rather both require zn . among group 

y pbgs orthologs , the e. coli enzyme requires catalytic 
 znb ( figure s1  ) while the other proteins do not require catalytic 
 divalent cations ( figure s1  ) .
the pattern of oligomeric states sampled by these orthologs is 
 also inconsistent , e.g. , dimer and octamer for p. aeruginosa(26 ) and t. gondii , while the e. coli(25 ) and y. enterocolitica proteins 
 ( e.k .
the v. cholerae pbgs samples another higher order multimeric 
 assembly in addition to the octamer ( e. k. jaffe , unpublished observation ) .
, several of 
 which are disease agents in humans . since the respective pbgs orthologs 
 have not been recombinantly produced , we were unable to include them 
 in our analysis of waladin activity
. therefore , we performed an in 
 silico analysis . a phylogenetic analysis ( maximum likelihood 
 method ) of the protein sequences of tested pbgs orthologs
inclusion of pbgs protein 
 sequences from pathogenic , obligate intracellular -proteobacteria rickettsia rickettsii and r. prowazekii ( figure s3 ) confirms their close relationship 
 to the wolbachia protein , suggesting that these orthologs 
 are susceptible to inhibition by waladin1 .
two members of obligate 
 intracellular bacteria of the chlamydiae family ( chlamydia 
 pneumonia and c. trachomatis ) are located 
 in a more isolated branch most closely related to the chloroplast p. sativum protein and the plastid - derived t. gondii ortholog ( figure s3 ) .
pbgs from both rickettsia and chlamydia are predicted 
 to be zn - independent and to possess an allosteric mg - binding site consistent with group x orthologs . as a 
 model for stimulated group y orthologs ,
the protein from p. 
 aerguinosa , one of the best characterized pbgs proteins , 
 was analyzed in more detail .
a buffer scan experiment was carried 
 out to monitor the influence of ph and concentrations of 5-ala and 
 kcl on the effect of waladin1 on papbgs ( figure s4 ) .
this experiment revealed that the 
 stimulatory effect of waladin1 that prevailed under standard conditions 
 ( figure 4a ) was 
 inhibitory in the absence of k , low ph , and low 5-ala 
 concentrations , ( figure 4b , figure s4 ) .
a similar buffer scan experiment for the other 
 group y orthologs from e. coli , v. cholerae , and y. enterocolitica did not reveal conditions 
 that resulted in inhibitory activity of the waladin1 ( parts a  c of figure s5 ) .
stimulatory and inhibitory 
 effects of waladin1 on papbgs . in a buffer scan experiment 
 testing various ph and 5-ala and 
 kcl concentrations (
all tested conditions are shown in figure s4 ) , experimental conditions were defined 
 under which waladin1 ( a ) stimulated papbgs activity 
 ( ph 8.0 , 5 mm 5-ala , 100 mm kcl ) or ( b ) inhibited papbgs activity ( ph 7.5 , 0.2 mm 5-ala , no kcl ) .
( c ) under the given 
 stimulatory conditions , increasing [ mg ] resulted in an 
 increased vmax in the presence of waladin1 . 
 the effect on the mgk0.5 was not consistent ( compare 100 to 200 m ) .
( d ) under inhibitory 
 conditions , the mg - response curve was shifted to the 
 right by increasing [ waladin1 ] .
curves were fit by nonlinear regression 
 assuming a sigmoidal ( four - parameter ) progression .
we aimed to determine whether the waladin1 activity 
 profile for papbgs under inhibitory conditions was 
 comparable to that 
 described above for stimulatory conditions ( table 2 ) or whether it was more similar to that of group x pbgs ( table 1 ) by determining the ic50 concentrations 
 for selected derivatives : waladin1 ( ic50 = 79  3 
 m ) ; 3 ( 94  4 m ) ; 5 ( no 
 inhibitory activity , highest concentration tested , = 533 m ) , 
 waladin2 ( 475  31 m ) .
although the compounds had a generally 
 weaker activity on papbgs ( under inhibitory conditions ) 
 compared to pspbgs and wpbgs , the 
 activity profile of waladins revealed key characteristics of group 
 x orthologs : dispensability of the r-2[(2-thienylcarbonyl)amino]ethyl ) 
 ( compare waladin1 to 3 ) , inactivity of the r - cooh positional isomer 5 , and the effectiveness of 
 waladin2 .
we proceeded to characterize the influence of the different buffer 
 components on papbgs activation and inhibition under 
  standard stimulatory 
( 100 mm tris - hcl , ph 8.0 , 1 mm 
 mgcl2 , 5 mm 5-ala , 100 mm kcl ) and  standard inhibitory  
 conditions ( 100 mm tris - hcl , ph 7.5 , 1 mm mgcl2 , 0.2 mm 
 5-ala , no kcl ) in more detail .
the inhibitory effect was similar 
 to that described for similar pspbgs and wpbgs . under 
conditions 
 waladin1 functionally competed with mg ( figure 4 d ) , but in contrast to group x pbgs ( parts c and d of figure s2 ) , maximum activity 
 of papbgs was restored at high mg ( 10 
 mm ) .
as papbgs does not require catalytic metal ions 
 but is allosterically activated by mg , this indicates that waladin1 reduces the affinity for the 
 allosteric mg or stabilizes a form of the protein with 
 a lower affinity for the allosteric mg ( e.g. , pro - octamer 
 dimer ) . under 
standard stimulatory  conditions the 
 affinity of papbgs for mg was not consistently 
 altered , but vmax was increased ( figure 4c ) .
monovalent k had been reported 
 to have an activating 
 effect on papbgs function and was included at high concentration in the buffer used for the 
 ec50 determinations described above .
k - dilution 
 series under  standard stimulatory  and  standard 
 inhibitory  conditions revealed that this monovalent cation 
 is required to achieve a stimulatory effect of waladin1 ( part a of figure s6 ) while antagonizing the inhibitory 
 effect ( part b of figure s6 ) . binding studies 
 using thermal shift assays
conducted in the presence and absence of 
 k showed that this ion had no influence on the binding 
 of waladin1 to papbgs ( parts 
 c and d of figure s6 ) .
we conclude that the conformational 
 changes of papbgs or the selection of certain assembly 
 states elicited by k - binding might alter the functional outcome of waladin1-binding .
classic 
 michaelis  menten kinetics demonstrated that under 
  standard stimulatory  conditions , km was not affected , while vmax was increased ( part a of figure s7 ) . 
 under 
conditions increasing [ waladin1 ] 
 led to a consistent increase in km , indicating 
 competition with 5-ala ; however , vmax was 
 only reduced at low inhibitor concentrations ( part b of figure s7 ) .
this pattern was also observed in the 
 initial buffer scan experiments ( figure s4 ) .
this noncanonical behavior might indicate binding of waladin1 
 with a different affinity and functional outcome to ( a ) different 
 binding sites or ( b ) the same binding site in different subpopulations 
 of protein ( e.g. , different oligomeric states or different protomers 
 of one oligomer ) .
pbgs is a morpheein and therefore can form a variety of different 
 oligomeric states including dimers , hexamers , or octamers specific 
 for each ortholog . to elucidate
whether 
 the inhibitory or stimulatory activity of waladin1 on pbgs proteins 
 is associated with stabilization of certain oligomer populations , 
 proteins were preincubated with a dilution series of waladin1 and 
 the oligomers were separated by nondenaturing page ( figure 5 ) .
activity of the protein oligomers in the gel 
 was measured by incubating the gel in an activity stain buffer that 
 included 5-ala , followed by in - gel staining for porphobilinogen production 
 with ehrlich s reagent .
waladin1 shifted the pspbgs oligomeric equilibrium away from the high - activity octameric 
 state to the low - activity hexameric assembly ( figure 5a ) and thus likely conferred inhibition .
porphobilinogen production 
 by pspbgs was not reduced in the in - gel activity 
 assay ( figure 5a , right panel ) , probably because 
 the excess of 5-ala in the buffer had displaced previously bound waladin1 
 from the protein ( according to the mixed inhibition model ) which then 
 diffused away from the protein reducing the local concentration .
5-ala 
 is known to induce a transition from hexameric or dimeric protein 
 to the active octamer for various pbgs orthologs .
therefore , the enzymatic activity of protein in the hexameric 
 band most likely resulted from a 5-ala - induced transition from hexameric 
 to octameric protein in the gel , as we have previously shown using 
 a 2d native gel technique .
waladin2 also led to 
 a stabilization of pspbgs hexamers , although with 
 weaker affinity than waladin1 ( data not shown ) .
( a ) 2 g of pspbgs , ( b , 
 f ) 3 g of papbgs , ( c ) 
 2 g of wpbgs , ( d ) 3 g of ecpbgs , or ( e ) 2 g vcpbgs and 2 g yepbgs were incubated for 60 min ( pspbgs ) 
 or 20 min ( all other proteins ) at 37 c in the respective optimal 
 buffer containing the indicated concentrations of k , mg , zn , 5-ala , and waladin1 before samples were 
 loaded onto a 7.5% nondenaturing page gel .
after gel electrophoreis , 
 an in - gel activity assay was conducted ( a , b , d , e ) .
protein bands 
 were stained with coomassie blue ( left panel ) , and porphobilinogen 
 production was detected with ehrlich s reagent ( right panel ) . 

( a ) incubation of pspbgs with 5 mm 5-ala for 60 min 
 shifted the oligomeric equilibrium from the hexamer toward the octamer . 

addition of waladin1 after 30 min followed by further preincubation 
 for 30 min reverted this transition and locked the protein in the 
 hexameric assembly .
( b ) in the presence of 1 mm mg and 
 100 mm k a large amount of papbgs assembled 
 into the octameric state , whereas incubation with waladin1 induced 
 enzymatically inactive dimers .
( c ) wpbgs was incubated 
 with different concentrations of dtt , mg , or 5-ala in 
 the absence / presence of waladin1 ( upper gel ) or waladin2 ( lower gel ) . 

waladin1 shifted the equilibrium away from octamer and induced all 
 dimeric , tetrameric , and hexameric assemblies , while waladin2 induced 
 the dimer only .
( d ) high concentrations of waladin1 ( 2 mm ) weakly 
 induced the low molecular weight ( putatively dimeric ) population of ecpbgs ( arrowheads ) .
( f ) papbgs was incubated for 30 min at 37 
 c in different ph buffers ( 100 mm tris - hcl , 100 mm kcl , ph 7.08.5 ) 
 in the presence / absence of 1 mm mg , 5 mm 5-ala , and waladin1 . 
 the octamer - inducing effect of 5 mm 5-ala dominated the dimer - inducing 
 effect of 250 m waladin1 .
preincubation with mg and k alone led to 
 a shift from the putative dimeric to the octameric assembly and induced 
 a population with high enzymatic activity ( figure 5b ) .
figure s8 shows a structural 
 representation of the papbgs octamer highlighting 
 the binding interfaces of both allosteric mg and k. in our native page assays , the octamer - inducing effect of 
 mg and k and the corresponding enzymatic 
 activity were abrogated by waladin1 in a concentration - dependent manner 
 ( figure 5b ) .
as the predominantly resulting 
 dimeric protein is expected to be enzymatically inactive , any porphobilinogen 
 production at this location , again , likely arose from in - gel transition 
 to octamers induced by 5-ala .
at waladin1 concentrations of 500 
 m all of the protein was present as a dimer ( figure 5b , left panel ) and had a decreased ability to produce 
 porphobilinogen as demonstrated by the in - gel assay ( figure 5b , right panel ) , indicating that the inhibitor successfully 
 prevented the 5-ala induced assembly of dimers into octamers during 
 the activity assay .
these results for papbgs and pspbgs demonstrated that waladin1 allosterically modulates 
 pbgs structure and , for these orthologs , locks the protein in its 
 inactive assembly state as the molecular basis for inhibition .
however , this mechanism is in disagreement with our previous observation 
 that waladin1 did not interfere with the oligomeric equilibrium of wpbgs but bound to and inhibited octameric protein . in those experiments
preincubation had been 
 performed in 1 protein loading buffer containing glycerol and 
 bromophenol blue that may have obscured an effect of waladin1 on the wpbgs oligomeric equilibrium .
we therefore reanalyzed the 
 effect of waladin1 on this endobacterial ortholog using the current 
 protocol where protein loading buffer was added after preincubation .
wpbgs was found to exist in an equilibrium of at least four 
 different assemblies including potential dimeric , tetrameric , hexameric , 
 and octameric states ( figure 5c , upper panel ) . 

the addition of dtt , mg , and 5-ala preferentially induced 
 the octamer , while waladin1 induced all the inactive lower molecular 
 weight assemblies .
while 5 mm 5-ala was still able to reverse the 
 effect of 25 m waladin1 , higher concentrations of waladin1 
 dominated the oligomeric equilibrium and prevented the octamer - inducing 
 effect of 5-ala .
in contrast to the benzimidazole waladin1 , the tricyclic 
 quinoline derivative waladin2 strongly induced the dimer but stabilized 
 neither tetramers nor hexamers ( figure 5c , 
 lower panel ) and was easily overcome by excess 5-ala .
thus , the inhibitory 
 mechanism for both waladin1 and waladin2 is also stabilization of 
 inactive assembly states of wpbgs . in analogy ,
the stimulatory effect by waladin1 observed for some 
 pbgs orthologs might be elicited by a stabilization of the active 
 octameric state and a concurrent increase in enzymatically active 
 protein .
however , native page experiments did not reveal evidence 
 for a waladin1-elicited stabilization of octamers for either of the 
 group y pbgs orthologs that might explain the stimulatory activity 
 of the compound ( figure 5b , d 
the highest waladin1 concentrations reduced the mg - stimulated conversion of dimer to octamer ( figure 5d , left panel ) , whereas neither the oligomeric equilibrium 
 of vcpbgs nor that of yepbgs was 
 markedly altered by preincubation with waladin1 ( figure 5e ) . as described above , we observed stabilization of papbgs dimers ( figure 5b ) under conditions 
 that , in the enzymatic assay , led to stimulation , not inhibition .
importantly , while present in the enzymatic assay , 5-ala was omitted 
 during the preincubation period for the native page experiments on papbgs shown in figure 5b .
we therefore 
 determined the effect of waladin1 on papbgs at different 
 ph values and in the presence and absence of 5 mm 5-ala ( figure 5f ) . while the octamer - stabilizing effect of mg and 5-ala and a ph dependency ( high ph favors octamers , 
 low ph favors hexamers ) were readily observed , no octamer - stabilizing 
 effect of waladin1 was discerned under any condition .
but while waladin1 
 was able to stabilize the papbgs dimer in the absence 
 of 5-ala , 5 mm 5-ala completely abrogated this effect ( figure 5 f ) , demonstrating that the status of the oligomeric 
 equilibrium ( as influenced by 5-ala and the ph ) dominates the outcome 
 of modulation by waladin1 .
as the percentage of enzymatic activation 
 achieved by waladin1 was generally very low ( 20% ) , it is uncertain 
 whether such minor differences would be properly reflected by a nonquantitative 
 method such as native page .
alternatively , it was possible that 
 the stimulation of catalytic 
 activity by waladin1 resulted from a mechanism increasing the catalytic 
 rate of pre - existing octamers rather than increasing the percentage 
 of octamers in the equilibrium
. in that instance the stimulatory effect 
 of walad1 should be additive to conditions that promote octamer formation . 

pbgs is known to exhibit a protein - concentration - dependent specific 
 activity with higher protein concentrations promoting octamer formation .
waladin1 dose  response curves in the presence of saturating 
 and nonsaturating papbgs concentrations revealed 
 that the specific activity of papbgs (  standard 
 stimulatory  conditions with 10 mm mgcl2 ) increased 
 with protein concentration until a maximum activity of (100 
 mol of porphobilinogen)h(mg 
 of protein ) was reached at 10 g / ml 
 protein ( figure 6a ) .
these specific activity 
 values were identical to those values achieved by stimulation of waladin1 
 at lower protein concentrations .
at saturating protein concentrations 
 with maximum specific activity , waladin1 had no additional stimulatory 
 properties ( figure 6a ) , which was confirmed 
 over the entire range in a mg - dilution series ( figure 6b ) .
similar findings were seen for vcpbgs , yepbgs , and ecpbgs ( data 
 not shown ) .
these data suggest that waladin1 and an increase in protein 
 concentration have functionally redundant effects in promoting an 
 active octameric assembly , although the final experimental proof of 
 octamer stabilization by waladin1 still has to be provided ( compare 
 figure 5d  f ) .
protein - concentration - dependent 
 specific activity of papbgs and waladin1 stimulation : 
 ( a ) waladin1 dose 
assay conditions 
 ( 100 mm tris - hcl , ph 8.0 , 10 mm mgcl2 , 100 mm kcl , 5 mm 
 5-ala ) ; ( b ) mg - response curve for 10 g / ml papbgs under the same conditions described in part a. the 
 stimulatory effect of waladin1 disappeared at this protein concentration .
products of the tetrapyrrole biosynthesis pathway fulfill essential 
 biological functions in nearly all organisms , and a blockade of this 
 pathway may provoke serious malfunction or death . because of the generally 
 high degree of conservation of the involved enzymes among different 
 species , this pathway has been difficult to exploit for the design 
 of antibiotics , antifungal , antiparasitic , herbicidal , or pest control 
 agents intended to specifically interfere with tetrapyrrole biosynthesis 
 of the target organisms without affecting that of humans . in 
 the current study we have tested the recently discovered class 
 of waladins , species - specific inhibitors of an endobacterial ( wolbachia of brugia malayi ) pbgs , against a variety of pbgs orthologs in in vitro 
 enzymatic inhibition tests to determine the potential effector spectrum 
 on different organisms . using the different orthologs , we found two 
 contrasting biological effects elicited by waladins , i.e. , inhibition 
 and stimulation .
the susceptibility to these two effects allows one 
 to assign the different pbgs orthologs into three categories : group 
 x pbgs orthologs are inhibited by waladin1 ; group y pbgs orthologs 
 are stimulated ; group z members are neither potently inhibited nor 
 stimulated .
it must be noted that inhibitory and stimulatory susceptibilities 
 are not mutually exclusive for a single protein , as demonstrated for 
 the p. aeruginosa enzyme , for which the position 
 of the oligomeric equilibrium determines whether waladins inhibit 
 or stimulate .
our results provide important insight into the 
 molecular mechanism 
 by which waladins act , as exemplified mostly for waladin1 .
different pbgs orthologs 
 are known to sample different oligomeric states , with the common feature 
 that equilibria of low and high activity assemblies exist .
the high activity states are usually octamers , 
 while dimers and hexamers have been described as the inactive assemblies 
 because they lack subunit interactions required to stabilize a closed 
 conformation of the active site lid .
the interconversion between hexamers 
 and octamers involves disassembly to two conformationally distinct 
 forms of dimers ( pro - octamer and pro - hexamer dimers ) with an intrinsic 
 propensity to assemble into octamers or hexamers , respectively . for 
 all inhibited group x pbgs orthologs we have demonstrated that waladin1 
 disturbs the oligomeric equilibrium of these proteins , thus stabilizing 
 the low activity hexamers ( pspbgs ) , dimers ( papbgs ) , or a variety of low molecular weight assemblies 
 ( wpbgs ) .
our previous finding that waladin1 had no 
 effect on the oligomeric equilibrium of wpbgs may be ascribed to the presence of 20% glyercol 
 in the preincubation buffer that may have led to artificial stabilization 
 of the octamer .
thus , waladin1 can be classified as a  morphlock  
 inhibitor that locks a morpheein in its inactive assembly state and 
 thus confers inhibition .
such molecules 
 have previously been detected by targeted in silico screening or screening in an in vitro gel 
 shift assay .
in contrast , waladin1 was 
 originally identified by an unbiased enzymatic assay screening for 
 inhibitors . this serendipitous
finding 
 of a morphlock inhibitor indicates the potential that oligomeric state 
 regulators of morpheeins have for inhibitor design .
not only 
 the sar profiles of pspbgs and wpbgs are highly analogous , but the proteins also share 
 a mixed competitive / noncompetitive mode of inhibition by waladin1 , 
 involving a functional competition with mg ( this study 
 and ref ( 21 ) ) .
this 
 mode of action may be explained using the morpheein model involving 
 regulation of the oligmeric equilibrium . both mg and 5-ala induce the active octameric form 
 and are thus in competition with waladin1 that stabilizes the inactive 
 assemblies .
it is interesting to note that waladin1 induced a wide 
 range of wpbgs oligomeric assemblies , whereas waladin2 
 only induced the dimer .
the differential stabilization pattern of 
 certain subsets of oligomers may explain the different results obtained 
 for waladin1 and waladin2 when determining the mode of inhibition 
 by enzyme kinetics .
in contrast to previously 
 described morphlocks , which inhibit by 
 stabilizing inactive pbgs assemblies , most stimulatory influence on the protein is mediated via an induction 
 of the highly enzymatically active octameric assembly , as is the case 
 for allosteric mg and k binding ( see structural 
 representation in figure s8 ) , active site 
 binding of 5-ala and high protein concentrations 
 ( a phenomenon known as protein concentration - dependent specific activity ) . in order to achieve a stimulatory effect
, waladin1 must bind to 
 and stabilize the octamers ( or pro - octamer dimers ) of group y pbgs 
 orthologs , and it is plausible that the stimulatory effect of waladin1 
 is also mediated by stabilization of octamers .
although we were not 
 able to directly demonstrate that stimulation of group y pbgs is mediated 
 via an induction of the octameric assembly in native page assays , 
 enzymatic assays revealed that waladin1-mediated stimulation was abolished 
 or strongly diminished at high protein concentrations .
this indicates 
 that waladin1 does not increase the catalytic rate of a single octamer 
 but rather modifies the oligomeric equilibrium in a way that promotes 
 the assembly of , and/or stabilizes , enzymatically active oligomers . 

this effect apparently becomes redundant with high pbgs concentration 
 when the octamer is strongly induced .
yet it remains to be determined 
 which equilibrium constants of the oligomerization pathway are affected 
 by waladin1 binding and how this effect differs from and integrates 
 with the octamer - inducing influence of mg or 5-ala .
the p. aeruginosa protein showed the peculiarity 
 that it was both inhibited and stimulated by waladin1 as a function 
 of ph , 5-ala , and kcl .
apparently , the modulator is able to bind both 
 to the dimeric and octameric assembly , which is likely to occur with 
 different affinities and different on- and off - rates . in the absence 
 of 5-ala ,
waladin1 drove the oligomeric equilibrium toward the inactive 
 dimeric state , whereas high concentrations of 5-ala abrogated this 
 effect , shifting the equilibrium toward the octamer .
thus , the overall 
 effect of waladin1-mediated modulation depends on the initial status 
 of the oligomeric equilibrium . in the absence of strong octamer - inducing 
 stimuli ( such as 5-ala ) the dimer - stabilizing effect prevails . in 
 contrast ,
when the dimer concentration falls below a certain threshold 
 ( e.g. , at high 5-ala , which is supported by high ph , mg , and k ) , waladin1 may bind predominantly to the octamer 
 ( or pro - octamer dimer or intermediates ) , temporarily stalling the 
 protein in this assembly and facilitating the formation of active 
 octamers which will become visible as a net stimulation effect .
this 
 model may explain the noncanonical progression of the substrate - response 
 curve ( figures s4 and s7 ) where , under 
 otherwise identical buffer conditions , the elevated 5-ala concentration 
 turned an inhibitory effect by waladin1 into stimulation . in 
 order to fully support the suggested modes of action a cocrystal 
 structure of a pbgs enzyme with a bound waladin modulator will be 
 required . to date
in contrast , for the p. aeruginosa protein and various group y 
 pbgs , like that from e. coli(24 ) and t. gondii , crystal 
 structures are available .
hence , solution of a crystal structure of 
 the p. aeruginosa pbgs with waladin1 bound may be 
 the most facile approach to initially determine the waladin binding 
 site .
all members of a pbgs group are inhibited / stimulated by a defined 
 chemotype of waladin1 benzimidazoles that share an sar profile , which 
 markedly differs from that of the other groups .
this is illustrated 
 best by focusing on the r-2[(2-thienylcarbonyl)amino]ethyl ) 
 substituent and its replacement by a hydrogen atom .
for group x orthologs 
 this alteration had a minor effect , but it abrogated activity on group 
 y , while it weakly increased activity on metazoan group z orthologs . 

a specific activity profile was also observed for the tricyclic quinoline 
 derivative waladin2 , which only inhibited members of the group x pbgs . 

interestingly , the activity profile of waladins for papbgs under stimulatory conditions was like that of other group y 
 pbgs ; however , under inhibitory conditions it was similar to that 
 of group x pbgs .
we therefore conclude that the difference between 
 the stimulatory and inhibitory outcome is not merely functional .
thus , 
 under the two conditions , structurally distinct waladin - binding sites 
 are exposed that have a different binding preference for a chemotype , 
 i.e. , for group x or group y pbgs .
these distinct binding environments 
 are likely related to certain states of the oligomeric equilibrium .
it is remarkable that the only two orthologs ( h. sapiens and d. melanogaster ) that did not feature an allosteric 
 mg - binding site were not affected by waladin1 , whereas 
 all mg - responsive orthologs were inhibited or stimulated . 
 beyond that , neither the requirement of pbgs enzymes for catalytic 
 metal ions nor the predicted oligomeric architectures of the orthologs 
 revealed an unambiguous pattern with respect to waladin1-responsiveness 
 and did not allow for a clear prediction of this property .
thus , based 
 on the activity spectrum presented in this study , phylogenetics ( figure s3 ) may be the most promising approach 
 for predicting the activity of waladins to pbgs of different species . 

the pbgs of t. gondii is targeted to the apicoplast , a nonphotosynthetic plastid found in apicomplexan 
 parasites that has been acquired through secondary endosymbiosis of 
 a red alga and is likely of cyanobacterial origin .
it is therefore interesting that tgpbgs responds 
 to waladin1 treatment similar to the -proteobacterial orthologs 
 and not to the chloroplast - derived pea enzyme .
a unique feature of tgpbgs is the presence of a c - terminal extension that stabilizes 
 the octameric assembly and does not sample the hexamer .
as waladin1 mediates inhibition of plastid pspbgs by stabilizing the hexamer , the absence of this assembly 
 from the tgpbgs oligomeric equilibrium may be an 
 explanation for the lack of inhibitory activity of waladins against 
 this apicomplexan pbgs .
the species - specific inhibition of waladins 
 on group x pbgs might 
 be exploited for drug development , provided that in vivo tests confirm 
 the efficacy of these heme biosynthesis inhibitors .
group x comprises 
 the wolbachia , p. sativum , and in 
 part the p. aeruginosa ortholog .
in addition to the 
 already demonstrated activity of waladin1 on filarial worms ( that 
 contain and are dependent upon wolbachia endosymbionts ) our phylogenetic analysis indicates that pbgs 
 from other intracellular -proteobacterial pathogens closely 
 related to wolbachia , such as rickettsia spp .
chlamydia(28 ) pbgs orthologs are most closely related to the chloroplast protein 
 of pea and the apicoplast protein of t. gondii , followed 
 by the wolbachia protein . in the absence of the c - terminal 
 extension such as that of the t. gondii ortholog 

that mediates octamer stabilization and may render the protein insusceptible 
 to waladin1-induced dimer - stabilization , it is likely that the chlamydia orthologs behave more like the p. sativum protein which is susceptible to inhibition by waladin1 via hexamer 
 stabilization .
confirmation of the antibiotic potential of waladins 
 against rickettsia and chlamydia ( causing , for example , typhus , rocky mountain spotted fever , and 
 sexually transmitted chlamydia ) would require purification of the 
 respective recombinant proteins and in vitro drug tests in the enzymatic 
 assay and bacterial culture and is outside the scope of this study . 

pbgs of photosynthetic eukaryotes such as p. sativum are targeted to the chloroplast and are phylogenetically related 
 as they are believed to have arisen from a common ancestor .
the inhibitory activity of waladins on the pea protein therefore 
 indicates that waladins might be potential herbicides . for the p. aeruginosa protein the major factor rendering
the enzyme 
 insusceptible to inhibition by waladin1 ( and instead promoting stimulation ) 
 is high substrate concentrations .
depending on the bacterial physiology , 
 it is possible that an inhibitory effect of waladin1 may occur in 
 vivo , which might affect bacterial viability or virulence . in the 
 setting of prolonged pbgs inhibition ,
it is possible that 5-ala may 
 eventually accumulate above the threshold to overcome the inhibition 
 ( turning the outcome to stimulation ) . yet because of pro - oxidative 
 effects , elevated 5-ala levels might have a toxic effect on the bacterium 
 similar to what we have observed previously for filarial nematode 
 in culture and as reported in the context 
 of various human disorders .
the 
 stimulatory activity of waladin1 and a number of its derivatives 
 on the pbgs enzymes from several -proteobacteria ( e. 
 coli , v. cholerae , y. enterocolitica ) and the apicomplexan parasite t. gondii adds another 
 layer of complexity ( and specificity ) to waladins . because of the 
 stimulatory effects on pbgs of these organisms , a potential therapeutic 
 use of waladin - like compounds might appear challenging , unless overproduction 
 of porphyrins and precursors has a ( photo)toxic effect on these organisms 
 as is observed with several forms of porphyria .
if future research is able to translate the concept of 
 pbgs stimulation also to the human enzyme , this might open new avenues 
 for the therapy of diseases characterized by reduced pbgs activity , 
 like lead poisoning , through ( partial ) rescue of the catalytic rate 
 of porphobilinogen formation via stimulation by waladin - like compounds .
the comprehensive analysis of the diverse biological effects of 
 waladin modulators on different pbgs orthologs , elucidation of the 
 corresponding chemical profiles of the small molecules , and mechanistic 
 insights presented in this work will provide the groundwork for the 
 development of tailored inhibitors or activators of heme biosynthesis 
 with potential therapeutic applications .
the vibrio cholerae ( vcpbgs , uniprotkb c3lpu7 ) , yersinia enterocolitica ( yepbgs , f4muj9 ) , escherichia coli ( ecpbgs , p0acb2 ) , drosophila melanogaster ( dmpbgs , q9vtv9 ) , and pisum sativum ( pspbgs , p30124 ) proteins were recombinantly expressed 
 in their native form without purification tags .
the general method 
 for protein purification was as follows ( details follow below ) .
bacterial 
 cells were disrupted , and the pbgs protein was precipitated using 
 ammonium sulfate .
the redissolved ammonium sulfate pellet was applied 
 to a phenyl sepharose - cl4b column that had been equilibrated in ammonium 
 sulfate / potassium phosphate . following a reverse ionic strength gradient , 
 pbgs protein eluted at 2 mm kpi , ph 7.0 .
the pool of active protein 
 was then applied to and further purified on a q - sepharose column using 
 a gradient from low to high ionic strength . in some cases
the q - sepharose 
 column separated different quaternary structure isoforms ( e.g. , octamer 
 and hexamer ) , and these isolated pools , though quite pure by sds  page , 
 may have undergone further purification on a sephacryl s-300 column 
 or a superdex 200 column .
column buffers generally contained the divalent 
 metal ions required / used by the various pbgs .
the full details of 
 the purification of pspbgs , dmpbgs , 
 and ecpbgs have been published .
vcpbgs , expressed 
 in e. coli from a pet3 vector construct , was purified 
 using a 2040% ammonium sulfate cut ; active protein was applied 
 to and purified on a phenyl sepharose column ( 4 c ; elution gradient 
 from 30 mm potassium phosphate , ph 7.5 , 15% ammonium sulfate to 2 
 mm potassium phosphate , ph 7.5 ) and a q - sepharose column ( room temperature ; 
 elution gradient from 30 mm tris - hcl , ph 7.0 , 1 mm mgcl2 to 30 mm tris - hcl , ph 7.0 , 10 mm mgcl2 , 0.4 m kcl ) .
all 
 buffers used for the phenyl sepharose and q - sepharose columns also 
 contained 10 mm 2-mercaptoethanol and 0.1 mm phenylmethylsulfonyl 
 fluoride .
the final step , a sephacryl s-300 column , was run in 0.1 
 m tris - hcl , ph 7 , 10 mm mgcl2 , 10 mm 2-mercaptoethanol .
yepbgs , was expressed in e. coli from a pet3 vector construct and purified using a 1530% 
 ammonium sulfate cut ; active protein was applied to and purified on 
 a phenyl sepharose column ( 4 c ; elution gradient from 30 mm 
 potassium phosphate , ph 7.5 , 5% ammonium sulfate to 2 mm potassium 
 phosphate , ph 7.5 ) and a q - sepharose column ( room temperature ; elution 
 gradient from 10 mm tris - hcl , ph 7.0 to 1 m tris - hcl , ph 7.0 ) .
as 
 above , all buffers used for the phenyl sepharose and q - sepharose columns 
 contained 10 mm 2-mercaptoethanol and 0.1 mm phenylmethylsulfonyl 
 fluoride .
wolbachia of brugia malayi pbgs 
 ( wpbgs , q5gsr3 ) was expressed with a c - terminal his - tag 
 as described . for recombinant expression of 
 the p. aeruginosa ortholog ( papbgs , 
 q59643 ) the hemb gene of p. aeruginosa strain pa01 ( kindly provided by dr .
b. henrichfreise , bonn , germany ) 
 was amplified by pcr with a primer set including ndei and xhoi restriction 
 sites in the forward and reverse primers , respectively : papbgs - ndei - fw_primer , 5-gtcatatgagcttcactcccgcc-3 ; papbgs - xhoi - rv_primer , 5-gtctcgagacgcccccgtcttaattgttct-3. all primers were 
 purchased from biolegio ( nijmegen , the netherlands ) .
pcr 
 reactions were performed using 500 nm primers , 200 m dntps , 
 0.02 u phusion taq ( new england biolabs , ipswich , ma , usa ) in 1 
 phusion buffer hf ( new england biolabs ) using the following amplification 
 protocol : initial denaturation at 98 c for 5 min followed by 
 35 amplification cycles ( 98 c for 10 s , 72 c for 80 s ) . 

after gel purification , the amplified dna fragment ( 1 kb ) 
 was cloned into the pet-21b vector ( merck chemicals , darmstadt , germany ) 
 encoding a his6-tag downstream of the xhoi restriction 
 site .
the expression plasmid was transformed into bl21 e. 
 coli ( life technologies , darmstadt , germany ) .
bacterial cultures 
 were grown at 37 c , 150 rpm until an od600 of 0.50.6 
 when protein expression was induced with 100 m isopropyl -d-1-thiogalactopyranoside ( iptg ; fermentas , st .
leon - rot , germany ) 
 for 24 h at room temperature , 150 rpm .
his - tagged wpbgs and papbgs were purified with ni - nta - agarose 
 ( qiagen , hilden , germany ) following the manufacturer s protocol .
wpbgs and papbgs were stored in 100 mm 
 tris - hcl , ph 8.0 , 300 mm nacl , 25 mm imidazole , 10% glycerol . the t. gondii hemb gene was cloned and expressed 
 as described by shanmugam et al . with 
 minor modifications using the following primers : tgpbgs - ndei - fw primer , 5-tatacatatgacgccacgggggcccctcgac-3 ; tgpbgs - bamhi - rv primer , 5-gatggatccttagtagcagggttctgtaaacttctgc-3. in 
 brief
, the amplified sequence was cloned into the pet-21b vector , 
 which was then transformed into bl21 e. coli .
bacterial 
 cultures were grown as above until od600 = 0.6 when protein 
 expression was induced with 100 m iptg overnight at 23 c , 
 180 rpm .
involving sonication , iterative 
 freeze  thaw cycles , an ammonium sulfate cut , and sequential 
 purification on a phenyl - sepharose cl-4b and a q sepharose high performance 
 column .
protein was finally eluted with gradient of 10 mm to 1 m tris - hcl , 
 ph 7.0 , 1 mm mgcl2 .
fractions containing active protein 
 were pooled , concentrated to 0.22 mg / ml , and stored at 80 
 c .
, reactions 
 were incubated in activity buffer ( see below ) with 5-ala ( sigma aldrich , 
 munich , germany ) at 36 c for 1060 min .
the reaction 
 was stopped by addition of 200 l of modified ehrlich s 
 reagent ( 1 g of 4-dimethylaminobenzaldehyde ( sigma , munich , 
 germany ) dissolved in 42 ml of acetic acid , 12 ml of perchloric acid , 
 and 7.3 ml of 12% trichloroacetic acid ) , incubated at room temperature 
 for 10 min , and the od555 was read .
ic50 determinations 
 of waladin1 and its derivatives for the different pbgs orthologs were 
 performed under buffer conditions optimal for each protein , all with 
 0.2 mm 5-ala .
these concentrations had previously been used in ic50 determinations for wpbgs , for which inhibitory 
 activity was dependent on mg and 5-ala concentration .
protein concentrations and reaction times were 
 adjusted to ensure a maximum substrate turnover of 30% .
this required 
 subsaturating pbgs concentrations at which protein - concentration - dependent 
 differences in the specific activity of the protein may prevail because 
 of the equilibrium of different quaternary assemblies of pbgs .
thus , 
 the standard reaction conditions for the different orthologs were 
 as follows .
all protein concentrations are listed as subunit concentrations 
 and specific activities under the given conditions are indicated in 
 brackets:(1)ecpbgs : 200 nm protein 
 in 100 mm bis - tris propane ( btp)-hcl , ph 8.1 , 1 mm mgcl2 , 10 m zncl2 , 5 mm dtt for 10 min [ (85.1 
 mol of porphobilinogen)/(mg of proteinh)](2)pspbgs : 300 nm protein 
 in 100 mm btp - hcl , ph 8.5 , 1 mm mgcl2 , 5 mm dtt for 15 
 min [ (11.4 mol of porphobilinogen)/(mg of proteinh)](3)vcpbgs : 
 125 nm protein 
 in 100 mm btp - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt , 100 mm 
 kcl for 10 min [ (32.7 mol of porphobilinogen)/(mg of 
 proteinh)](4)yepbgs : 150 nm protein 
 in 100 mm btp - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt , 100 mm 
 kcl for 10 min [ (25.7 mol of porphobilinogen)/(mg of 
 proteinh)](5)papbgs : 150 nm protein 
 in 100 mm tris - hcl , ph 8.0 , 1 mm mgcl2 , 100 mm kcl for 
 10 min [ (19.3 mol of porphobilinogen)/(mg of proteinh)](6)tgpbgs : 
 250 nm protein 
 in 100 mm tris - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt for 20 
 min [ (8.9 mol of porphobilinogen)/(mg of proteinh)](7)dmpbgs : 
 400 nm protein 
 in 100 mm btp - hcl ph 8.0 , 0.5 m zncl2 , 100 m 
 dtt for 60 min . [
1.0 mol porphobilinogen/(mg protein 
 * hour ) ] ecpbgs : 200 nm protein 
 in 100 mm bis - tris propane ( btp)-hcl , ph 8.1 , 1 mm mgcl2 , 10 m zncl2 , 5 mm dtt for 10 min [ (85.1 
 mol of porphobilinogen)/(mg of proteinh ) ] pspbgs : 300 nm protein 
 in 100 mm btp - hcl , ph 8.5 , 1 mm mgcl2 , 5 mm dtt for 15 
 min [ (11.4 mol of porphobilinogen)/(mg of proteinh ) ] vcpbgs : 
 125 nm protein 
 in 100 mm btp - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt , 100 mm 
 kcl for 10 min [ (32.7 mol of porphobilinogen)/(mg of 
 proteinh ) ] yepbgs : 150 nm protein 
 in 100 mm btp - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt , 100 mm 
 kcl for 10 min [ (25.7 mol of porphobilinogen)/(mg of 
 proteinh ) ] papbgs : 150 nm protein 
 in 100 mm tris - hcl , ph 8.0 , 1 mm mgcl2 , 100 mm kcl for 
 10 min [ (19.3 mol of porphobilinogen)/(mg of proteinh ) ] tgpbgs : 
 250 nm protein 
 in 100 mm tris - hcl , ph 8.0 , 1 mm mgcl2 , 5 mm dtt for 20 
 min [ (8.9 mol of porphobilinogen)/(mg of proteinh ) ] dmpbgs : 
 400 nm protein 
 in 100 mm btp - hcl ph 8.0 , 0.5 m zncl2 , 100 m 
 dtt for 60 min . [ 1.0 mol porphobilinogen/(mg protein 
 * hour ) ] benzimidazole 
 compounds 114 were synthesized as 
 described previously .
the purity 
 of the compounds was determined on an agilent 1100 series hplc ( agilent , 
 germany ) using a c18 eclipse xdb - c18 column ( 4.6 mm  150 mm , 
 agilent ) by applying a gradient of doubly distilled h2o 
 up to 100% acetonitrile in 25 min ( flow rate , 1 ml / min ) .
pbgs proteins 
 were preincubated 
 for 10 min at 37 c in their respective enzymatic assay buffer 
 ( see above ) containing different concentrations of mg , k , 5-ala , zn , and waladin1 .
samples were 
 then put on ice , and 2 protein loading buffer was added ( 0.2 
 m tris - hcl , ph 8.8 , 40% glycerol , 0.005% bromophenol blue ) . an amount 
 of 23 g of protein was loaded onto a 7.5% nondenaturing 
 page gel ( 1 gel buffer , 7 mm tris - hcl , 7 mm sodium acetate , 
 ph 6.5 ) and run at 2025 v , 25 ma , 4 c for 1520 
 h ( running buffer , 25 mm tris - hcl , ph 8.8 , 80 mm glycine ) . for monitoring 
 of in - gel enzymatic activity , the gels were soaked in activity stain 
 buffer ( see below ) , incubated for 1015 min at 36 c and 
 porphobilinogen was stained with modified ehrlich s reagent 
 ( see above ) .
gels were scanned on a flatbed scanner , quickly rinsed 
 in h2o and stained with coomassie blue for 20 min , destained 
 with 30% methanol for 2 h to overnight , and scanned again .
activity 
 stain buffer composition was as follows : papbgs , 
 100 mm tris - hcl ( ph 8.0 ) , 100 mm kcl , 1 mm mgcl2 , 500 m 
 5-ala ; pspbgs , 100 mm btp - hcl ( ph 8.5 ) , 1 mm mgcl2 , 100 m dtt , 500 m 5-ala ; ecpbgs , 100 mm btp ( ph 8.1 ) , 1 mm mgcl2 , 100 m zncl2 , 100 m dtt , 500 m 5-ala ; vcpbg / yepbgs , 100 mm btp - hcl ( ph 8.0 ) , 100 mm kcl , 
 10 mm mgcl2 , 100 m dtt , 500 m 5-ala .
data were analyzed with 
 prism 5.02 for windows ( graphpad software , san diego , ca , usa ) .
half 
 maximal inhibitory concentration values ( ic50 ) were determined 
 by fitting logarithmical data to the  log(inhibitor ) vs normalized 
 response  variable slope 
algorithm [ y = 100/(1 + 10 ) ] with h as the hill 
 coefficient .
half maximal effective concentration values for stimulatory 
 effects (  ec50  stimulation induced by waladins 
 on several pbgs orthologs ) were fit with the  log(agonist ) 
 vs response  variable slope  algorithm [ y = bottom + ( top  bottom)/(1 + 10 ) ] .
the binding 
 constant for mg ( k0.5 ) was 
 approximated from activity data using the same equation .
data from 
 substrate concentration series were fit individually by nonlinear 
 regression assuming conventional michaelis 
menten kinetics 
 [ y = vmaxx/(km + x ) ] , km being the michaelis  menten constant 
 and vmax being the maximum enzymatic velocity . 

eadie  hofstee transformation followed by linear regression 
 was performed for a linearized representation of these data .
if not 
 stated otherwise , graphs show the mean  sd from triplicate measurements .
figure s8 was prepared from the protein data 
 bank file 1gzg ( http://www.rcsb.org/pdb/explore.do?structureid=1gzg ) 
 using the pymol molecular graphics system , version 1.3 , from schrdinger , 
 llc .
 the 
 heme biosynthesis enzyme porphobilinogen synthase ( pbgs ) is 
 a potential drug target in several human pathogens . waladin1 
 benzimidazoles 
 have emerged as species - selective pbgs inhibitors against wolbachia endobacteria of filarial worms . in the present 
 study , we have systematically tested waladins against pbgs orthologs 
 from bacteria , protozoa , metazoa , and plants to elucidate the inhibitory 
 spectrum . 
 however , the effect of waladin1 on different pbgs orthologs 
 was not limited to inhibition : several orthologs were stimulated by 
 waladin1 ; others remained unaffected . 
 we demonstrate that waladins 
 allosterically modulate the pbgs homooligomeric equilibrium with inhibition 
 mediated by favoring low - activity oligomers , while 5-aminolevulinic 
 acid , mg2 + , or k+ stabilized high - activity oligomers . 
 pseudomonas aeruginosa pbgs could be inhibited or stimulated 
 by waladin1 depending on these factors and ph . 
 we have defined the 
 waladin chemotypes responsible for either inhibition or stimulation , 
 facilitating the design of tailored pbgs modulators for potential 
 application as antimicrobial agents , herbicides , or drugs for porphyric 
 disorders . 
Introduction
Results
Discussion and Conclusions
Experimental
Section