Literature summary for 1.1.1.205 extracted from

Makowska-Grzyska, M.; Kim, Y.; Gorla, S.K.; Wei, Y.; Mandapati, K.; Zhang, M.; Maltseva, N.; Modi, G.; Boshoff, H.I.; Gu, M.; Aldrich, C.; Cuny, G.D.; Hedstrom, L.; Joachimiak, A.
Mycobacterium tuberculosis IMPDH in complexes with substrates, products and antitubercular compounds (2015), PLoS ONE, 10, e0138976.

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Application

Application	Comment	Organism
drug development	inosine 5'-monophosphate dehydrogenase 2 (IMPDH2) from Mycobacterium tuberculosis is an attractive drug target	Mycobacterium tuberculosis

Cloned(Commentary)

Cloned (Comment)	Organism
gene guaB2, recombinant expression of wild-type and mutant enzymes in Escherichia coli strain BL21(DE3)	Mycobacterium tuberculosis

Crystallization (Commentary)

Crystallization (Comment)	Organism
purified recombinant MtbIMPDH2DELTACBS enzyme mutant in apofomr or complexed with IMP/NAD+, or with inhibitors MAD1/IMP, P41/IMP, or Q67/IMP, the crystallization conditions are for the apoform mutant 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, for the MAD1/IMP-enzyme mutant complex 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, for the P41/IMP-enzyme mutant complex 0.3 M magnesium formate, 0.1 M Tris, pH 8.5, 16°C, for the Q67/IMP-enzyme mutant complex 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, and for the NAD+/IMP-enzyme mutant complex 0.4 M magnesium formate, 0.1M Tris-HCl, pH 8.5, 20% sucrose, 16°C, crystals are soaked with 200 mM ligand solutions, X-ray diffraction structure determination and analysis at 1.60-2.00 A resolution. Analysis of the crystal structure of the enzyme mutant in complex with XMP and NAD+, detailed overview	Mycobacterium tuberculosis

Crystallization (Comment)

Organism

purified recombinant MtbIMPDH2DELTACBS enzyme mutant in apofomr or complexed with IMP/NAD+, or with inhibitors MAD1/IMP, P41/IMP, or Q67/IMP, the crystallization conditions are for the apoform mutant 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, for the MAD1/IMP-enzyme mutant complex 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, for the P41/IMP-enzyme mutant complex 0.3 M magnesium formate, 0.1 M Tris, pH 8.5, 16°C, for the Q67/IMP-enzyme mutant complex 0.1 M sodium/potassium phosphate, pH 6.2, 25% 1,2-propandiol, 10% glycerol, 16°C, and for the NAD+/IMP-enzyme mutant complex 0.4 M magnesium formate, 0.1M Tris-HCl, pH 8.5, 20% sucrose, 16°C, crystals are soaked with 200 mM ligand solutions, X-ray diffraction structure determination and analysis at 1.60-2.00 A resolution. Analysis of the crystal structure of the enzyme mutant in complex with XMP and NAD+, detailed overview

Mycobacterium tuberculosis

Protein Variants

Protein Variants	Comment	Organism
additional information	deletion of IMPDH CBS domain of MtbIMPDH2, residues E126-R252 are replaced with a GG linker. The MtbIMPDH2DELTACBS mutant shows significantly improved solubility and crystallizability properties compared to the wild-type enzyme, with the steady state kinetic parameters comparable to those reported for the wild-type enzyme	Mycobacterium tuberculosis

Inhibitors

Inhibitors	Comment	Organism
(2R)-2-phenyl-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
(2S)-2-(2,3-dichlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
(2S)-2-(2,3-dichlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-6-yl]propanamide	-	Mycobacterium tuberculosis
(2S)-2-(2,3-dimethoxyphenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
(2S)-2-(2-chloro-3-nitrophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
(2S)-2-(2-chlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
2-(2,4-dichlorophenoxy)-N-[2-(pyridin-2-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
2-(2-chlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
2-(4-methoxyphenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
2-chloro-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]-N,N-dimethylbenzamide	-	Mycobacterium tuberculosis
2-chloro-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]benzamide	-	Mycobacterium tuberculosis
2-chloro-N,N-diethyl-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]benzamide	-	Mycobacterium tuberculosis
2-chloro-N,N-dimethyl-5-[([2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl]carbamoyl)amino]benzamide	noncompetitive inhibitor, that has similar affinity to both E-IMP and E-XMP, enzyme binding structure analysis, overview	Mycobacterium tuberculosis
2-phenoxy-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	-	Mycobacterium tuberculosis
5'-O-([1-[(2E)-4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-2-methylbut-2-en-1-yl]-1H-1,2,3-triazol-4-yl]methyl)adenosine	an uncompetitive inhibitor with a strong preference for the E-XMP reaction intermediate, enzyme binding structure analysis, overview	Mycobacterium tuberculosis
additional information	IMPDH has multiple active site states that can be targeted for inhibitor design, and both the IMP/XMP and cofactor binding sites are exploited for that purpose, inhibitor screening of compounds with antitubercular activity, overview. Mechanism of MtbIMPDH2DELTACBS enzyme mutant inhibition	Mycobacterium tuberculosis
N-[4-chloro-3-(morpholine-4-carbonyl)phenyl]-N'-(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)urea	-	Mycobacterium tuberculosis
N2-(2,3-dichlorophenyl)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]-L-alaninamide	a competitive inhibitor versus NAD+, that has a strong preference for E-IMP, enzyme binding structure analysis, overview	Mycobacterium tuberculosis
NAD+	low substrate inhibition	Mycobacterium tuberculosis
N~2~-(2,3-dichlorophenyl)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]alaninamide	-	Mycobacterium tuberculosis

KM Value [mM]

KM Value [mM]	KM Value Maximum [mM]	Substrate	Comment	Organism
0.041	-	IMP	recombinant MtbIMPDH2DELTACBS mutant enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
0.078	-	IMP	recombinant wild-type enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
0.58	-	NAD+	recombinant MtbIMPDH2DELTACBS mutant enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
1.005	-	NAD+	recombinant wild-type enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis

Natural Substrates/ Products (Substrates)

Natural Substrates	Organism	Comment (Nat. Sub.)	Natural Products	Comment (Nat. Pro.)	Rev.	Reac.
IMP + NAD+ + H2O	Mycobacterium tuberculosis	-	XMP + NADH + H+	-	?
IMP + NAD+ + H2O	Mycobacterium tuberculosis H37Rv	-	XMP + NADH + H+	-	?

Organism

Organism	UniProt	Comment	Textmining
Mycobacterium tuberculosis	P9WKI7	-	-
Mycobacterium tuberculosis H37Rv	P9WKI7	-	-

Purification (Commentary)

Purification (Comment)	Organism
recombinant wild-type and mutant enzymes from Escherichia coli strain Bl21(DE3)	Mycobacterium tuberculosis

Reaction

Reaction	Comment	Organism	Reaction ID
IMP + NAD+ + H2O = XMP + NADH + H+	the enzymatic mechanism of IMPDH consists two steps, a dehydrogenase and a hydrolase reaction. Upon binding of IMP and NAD+ cofactor, a thioimidate enzyme-substrate adduct, E-XMP, is formed via a covalent bond to the catalytic C341 with concurrent production of NADH. Hydride transfer occurs to the pro-S position with the cofactor in the anti-conformation. The cofactor is released and an active site mobile flap moves into the NAD+ site and facilitates E-XMP hydrolysis with a conserved R443 acting as a general base. The enzyme has two distinct conformations, an open form for the dehydrogenase reaction and a closed form for E-XMP* hydrolysis	Mycobacterium tuberculosis	a

Substrates and Products (Substrate)

Substrates	Comment Substrates	Organism	Products	Comment (Products)	Rev.	Reac.
IMP + NAD+ + H2O	-	Mycobacterium tuberculosis	XMP + NADH + H+	-	?
IMP + NAD+ + H2O	-	Mycobacterium tuberculosis H37Rv	XMP + NADH + H+	-	?

Synonyms

Synonyms	Comment	Organism
guaB2	-	Mycobacterium tuberculosis
IMPDH2	-	Mycobacterium tuberculosis
inosine 5-monophosphate dehydrogenase 2	-	Mycobacterium tuberculosis

Temperature Optimum [°C]

Temperature Optimum [°C]	Temperature Optimum Maximum [°C]	Comment	Organism
22	-	assay at room temperature	Mycobacterium tuberculosis

Turnover Number [1/s]

Turnover Number Minimum [1/s]	Turnover Number Maximum [1/s]	Substrate	Comment	Organism
0.53	-	NAD+	recombinant wild-type enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
0.53	-	IMP	recombinant wild-type enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
0.57	-	NAD+	recombinant MtbIMPDH2DELTACBS mutant enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
0.57	-	IMP	recombinant MtbIMPDH2DELTACBS mutant enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis

pH Optimum

pH Optimum Minimum	pH Optimum Maximum	Comment	Organism
8	-	assay at	Mycobacterium tuberculosis

Cofactor

Cofactor	Comment	Organism	Structure
NAD+	dependent on, binding structure to CBS domain-deleted enzyme mutant of MtbIMPDH2DELTACBS	Mycobacterium tuberculosis

Ki Value [mM]

Ki Value [mM]	Ki Value maximum [mM]	Inhibitor	Comment	Organism
0.000013	-	2-chloro-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]-N,N-dimethylbenzamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000014	-	N2-(2,3-dichlorophenyl)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]-L-alaninamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000017	-	2-chloro-N,N-dimethyl-5-[([2-[3-(prop-1-en-2-yl)phenyl]propan-2-yl]carbamoyl)amino]benzamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000035	-	2-chloro-N,N-diethyl-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]benzamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000037	-	N-[4-chloro-3-(morpholine-4-carbonyl)phenyl]-N'-(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)urea	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00004	-	(2S)-2-(2,3-dichlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-6-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00004	-	N~2~-(2,3-dichlorophenyl)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]alaninamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000076	-	(2S)-2-(2,3-dichlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.0001	-	(2S)-2-(2-chlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00013	-	(2S)-2-(2-chloro-3-nitrophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00015	-	2-(4-methoxyphenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.000158	-	2-chloro-5-[[(2-[3-[(1Z)-N-hydroxyethanimidoyl]phenyl]propan-2-yl)carbamoyl]amino]benzamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00024	-	2-(2-chlorophenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00044	-	(2R)-2-phenyl-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00062	-	(2S)-2-(2,3-dimethoxyphenoxy)-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00065	-	2-phenoxy-N-[2-(pyridin-4-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
0.00158	-	5'-O-([1-[(2E)-4-(4-hydroxy-6-methoxy-7-methyl-3-oxo-1,3-dihydro-2-benzofuran-5-yl)-2-methylbut-2-en-1-yl]-1H-1,2,3-triazol-4-yl]methyl)adenosine	pH 8.0, 22°C	Mycobacterium tuberculosis
0.002	-	2-(2,4-dichlorophenoxy)-N-[2-(pyridin-2-yl)-1,3-benzoxazol-5-yl]propanamide	pH 8.0, 22°C	Mycobacterium tuberculosis
5	-	NAD+	recombinant wild-type enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis
16	-	NAD+	recombinant MtbIMPDH2DELTACBS mutant enzyme, pH 8.0, 22°C	Mycobacterium tuberculosis

General Information

General Information	Comment	Organism
additional information	determination of XMP and NAD+ substrate binding structures, overview. Active site structures of wild-type apo-enzyme and of CBS domain-deleted enzyme mutant MtbIMPDH2DELTACBS in apoform	Mycobacterium tuberculosis
physiological function	the enzyme IMPDH2 catalyzes the conversion of inosine 5'-monophosphate into xanthosine 5'-monophosphate with the concomitant reduction of NAD+ to NADH and controls flux into the guanine nucleotide pool	Mycobacterium tuberculosis