The non-mevalonate isoprenoid biosynthetic pathway as antiinfective drug target. Substrate synthesis and high throughput screening methods


 S1. Bacterial strains and plasmids used in this study 5. Table S2. Oligonucleotides used in this study 6. Table S3. List of recombinant proteins used for screening, substrate synthesis and as U ml -1 ), pyruvate kinase (12.5 U ml -1 ) and CMP kinase (6.25 U ml -1 ) in 112.5 mM Tris hydrochloride, pH 8.0 were added to 20 µl of a soution of 10 % (v/v) DMSO that may or may not contain a predertimined amount of a test compound in one well of a 96-well plate. The reaction was started by adding 80 µl of a solution containing 11.25 mM MgCl 2 , 1.125 mM dithiothreitol and IspF S4 protein (0.03 U ml -1 ) in 112.5 mM Tris hydrochloride, pH 8.0. The mixture was incubated at 27 °C, and the reaction was monitored photometrically at 340 nm. NMR spectroscopy. 1 H NMR and 1 H-decoupled 13 C NMR spectra were recorded at 500.1 and 125.5 MHz for 1 H and 13 C, respectively. The chemical shifts were referenced to external trimethyl silylpropane sulfonate. 31 



  photometrically at 340 nm. 


Table of Content: 1. Materials 2. High-throughput screens for IspC, IspD, IspE and IspF 3. NMR spectroscopy 4. Table

  

Table S1 .

 S1 P NMR spectra were recorded at a frequency of 101.3 MHz. 31 P chemical shifts were referenced to external 85 % H 3 PO 4 . Samples were dissolved in 150 mM Tris Bacterial strains and plasmids used in this study 


	Strain or plasmid Genotype or relevant characteristic	Reference
	E. coli		
	XL1-Blue	RecA1, endA1, gyrA96, thi-1, hsdR17, supE44, relA1, lac, [F´, proAB, lacI q Z∆M15, Tn10 (tet r )]	6
	M15 [pREP4]	Lac, ara, gal, mtl, recA + , uvr + , [pREP4, lacI, kan r ]	7
	Plasmids		
	pQE30	High copy N-terminal His-tag vector	8
	pQEfbA	Hyperexpression of fructose 1,6-bisphosphate aldolase from E. coli	this study
	hydrochloride, pH 8.0. pQEpfkA Hyperexpression of phosphofructokinase from E. coli	this study
	pQEpgi	Hyperexpression of glucose 6-phosphate isomerase from E. coli	this study
	pQEcmpK	Hyperexpression of CMP kinase from E. coli	this study
		S5	
		S6	

Table S2 .

 S2 Oligonucleotides used in this study 


	Designation	5'-Sequence-3'
	fbABamHIvo	GGAATCGGATCCATGTCTAAGATTTTTGATTTCG
	fbASalIhi	GGTAGCGTCGACTTACAGAACGTCGATCGCG
	pfkABamHIvo CCTTGAGGATCCATGATTAAGAAAATCGGTGTGTTG
	pfkAPstIhi	CCAATCCTGCAGATACAGTTTTTTCGCGCAGTCC
	pgiBamHIvo	AGAGTAGGATCCATGAAAAACATCAATCCAACGC
	pgiPstIhi	TATCGACTGCAGTTAACCGCGCCACGC
	CMPKvo	CCTGACGGATCCATGACGGCAATTGCCCCGG
	CMPKhi	CTGACCCTGCAGTTATGCGAGAGCCAATTTCTGG

Recognition sites for the restriction endonucleases BamHI, PstI and SalI are underlined S7 
Table S3 .

 S3 List of recombinant proteins used for screening, substrate synthesis and as auxiliary enzymes in enzyme assays 


	Protein	Source	Reference
	Dxs	Bacillus subtilis	1
	IspC	Escherichia coli	2
	IspD	Escherichia coli	9
	IspE	Escherichia coli	4
	PfkA	Escherichia coli	this study
	FbA	Escherichia coli	this study
	Pgi	Escherichia coli	this study
	CmpK	Escherichia coli	this study
Victoria Illarionova, Johannes Kaiser, Elena Ostrozhenkova, Adelbert Bacher, Markus Fischer, Wolfgang Eisenreich, Felix Rohdich
The non-mevalonate isoprenoid biosynthetic pathway as antiinfective drug target. Substrate synthesis and high throughput screening methods