Substituting the catalytic proline of 4-oxalocrotonate tautomerase with non-canonical analogues reveals a finely tuned catalytic system


Supplementary Figures and Tables

Supplementary Tables Table S1. Data collection and refinement statistics for 4-OT-9 (PDB entry 6GHW). Statistics for the highest-resolution shell are shown in parentheses. 

Figure S1 .Figure S2 .Figure S4 .

 S1S2S4 Figure S1. Growth of the proline auxotrophic Escherichia coli strain expressing 4-OT in a typical incorporation experiment. The strain was grown in M9 minimal medium containing a limited amount of proline until the depletion of the available proline. The analogue was then added, and the expression of 4-OT was induced. The apparent increase in D600 in the culture containing the analogue is most likely caused by the accumulation of inclusion bodies during overexpression since some of the 4-OT ended up in inclusion bodies according to SDS-PAGE. 


Table S2 .

 S2 Primers used in this study. 


	Primer	Sequence 5'3'
	4OT_Fwd	CAATTTCACACAGAATTCATTAAAGAGGAGAAATTAAGCATGCCTATTGCCCAGATCCAC
	4OT_Rev	CTATCAACAGGAGTCCAAGCTCAGCTAATTAAGCTTGGCTGCAGTTATCAGCGTCTGACCTTGCTGGCC
	MAP_Fwd	AATTGGTACCGGAAA AAGGAGATCTGCATATGGCTATCTCAATCAAGACCC
	MAP_Rev ProS_Fwd	CCATTAATTAATTATTCGTCGTGCGAGAT TATC GCC 4-OT-9 AATTG CATGCGTACTAGCCAATACCTGCTCTCCA
	Wavelength ProS_Rev	0.8731 TTAAAAGCTTTTATTAGCCTTTAATCTGTTTCACCA GATATTCGACG
	Resolution range ProS_G_Fwd	34.4 -2.3 (2.38 -2.3) GATAATCTCGCACGACGAATAATTAATGAATTCATTAAAGAGGAGAAATTAAGCATGCGT
	Space group ProS_G_Rev	R 3 2 TTCTGCGTTCTGATTTAATCTTAATTTATTAGCCTTTAATCTGTTTCACCAGATATTCGACG
	F1 Fwd Unit cell (Å, °) F1 Rev	85.30, 85.30, 155.46 GCGCTAGCGGAGTGTATACTGG 90, 90, 120 ATTCTAGAGACCCGTCGACCATCAGGCATCAAATAAAACG
	Total reflections F2 Fwd	26815 (1899) CTGATGGTCGACGGGTCTCTAGAATTTAAATGCGACAATTAATCA
	Unique reflections F2 Rev	9550 (764) TTGAGATAGCCATTAGTATATCTCCTTGAATTCGTTTAGTTCC
	Multiplicity F3 Fwd	2.8 (2.5) GGAGATATACTAATGGCTATCTCAATCAAGACCCC
	Completeness (%) F3 Rev	95.99 (78.26) GTAAGCCAGTATACACTCCGCTAGCGCTAATTCTCATGTTTGACAGCTTATCCAAAAAGG
	Mean I/sigma(I) 4OT34ProNNK_Fwd CCCTGGATGCGNNKCTGACCAG 4.55 (1.53)
	Wilson B-factor 4OT34ProNNK_Rev CAGMNNCGCATCCAGGGAGCGC 27.37
	R-merge 4OTGibson_Fwd	0.162 (0.541) GTGAGCGGATAACAATTTCACACAGAATTC
	R-meas 4OTGibson_Rev	0.199 (0.680) ACAGGAGTCCAAGCTCAGCTAATTAAGCTTG
	R-pim		0.114 (0.405)
	CC1/2		0.97 (0.62)
	CC*		0.99 (0.87)
	Reflections used in refinement	9548 (763)
	Reflections used for R-free	494 (43)
	R-work		0.289 (0.336)
	R-free		0.319 (0.367)
	CC(work)		0.886 (0.620)
	CC(free)		0.837 (0.717)
	Number of non-hydrogen atoms	1368
	macromolecules		1299
	ligands		1
	solvent		68
	Protein residues		171
	RMS(bonds)		0.003
	RMS(angles)		0.46
	Ramachandran favored (%)	100.00
	Ramachandran allowed (%)	0.00
	Ramachandran outliers (%)	0.00
	Rotamer outliers (%)	1.48
	Clashscore		2.67
	Average B-factor		30.28
	macromolecules		30.13
	ligands		45.92
	solvent		32.84
			SI page 9
Michael S Lukesch, Tea Pavkov-Keller, Karl Gruber, Klaus Zangger, Birgit Wiltschi
Substituting the catalytic proline of 4-oxalocrotonate tautomerase with non-canonical analogues reveals a finely tuned catalytic system