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Information on EC 5.1.1.1 - alanine racemase and Organism(s) Mycobacterium tuberculosis and UniProt Accession P9WQA9
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5.1.1.1 alanine racemaseIUBMB Comments
A pyridoxal-phosphate protein.
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Word Map
- 5.1.1.1
- pyridoxal
- 5'-phosphate
- peptidoglycan
-
racemization
- d-cycloserine
- stearothermophilus
-
plp-dependent
-
d-amino
-
aldimine
- d-alanyl-d-alanine
- exosporium
-
pyridoxal-5'-phosphate-dependent
- drug development
-
5'-phosphate-dependent
-
d-alanine:d-alanine
- medicine
- biotechnology
- pharmacology
The taxonomic range for the selected organisms is: Mycobacterium tuberculosis
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
The expected taxonomic range for this enzyme is: Bacteria, Archaea, Eukaryota
Synonyms
alanine racemase, alr-2, d-alanine racemase, alrbax, mbalr2, alrtt, cdalr, l-alanine racemase, alraba, cbl/alr, 
more
topSYNONYM 
ORGANISM
UNIPROT
COMMENTARY 
LITERATURE
L-Alanine racemase
-
-
-
-
L-Alanine:D-alanine racemase
-
-
-
-
Racemase, alanine
-
-
-
-
REACTION TYPE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
racemization
-
-
-
-
SYSTEMATIC NAME
IUBMB Comments
alanine racemase
A pyridoxal-phosphate protein.
CAS REGISTRY NUMBER
COMMENTARY
9024-06-0
-
SUBSTRATE
PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
Reversibility
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
NATURAL SUBSTRATE
NATURAL PRODUCT
REACTION DIAGRAM
ORGANISM
UNIPROT
COMMENTARY
(Substrate)
(Substrate)
LITERATURE
(Substrate)
(Substrate)
COMMENTARY
(Product)
(Product)
LITERATURE
(Product)
(Product)
REVERSIBILITY
r=reversible
ir=irreversible
?=not specified
r=reversible
ir=irreversible
?=not specified
COFACTOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
cycloserine
D-cycloserine or a racemic mixture of D- and L-cycloserine
D-cycloserine
specific inhibition is reversible by D-alanine in the growth medium
(2S)-1-oxo-1-([(1R)-1-phosphonoethyl]amino)propan-2-yl L-methioninate
-
-
1,1'-(2-oxido-1,2,5-oxadiazole-3,4-diyl)-bis (1-(2-thienyl))-methanone
-
-
2-(2-chloro-4-nitrophenyl)-4-(2,3-dihydro-1H-inden-2-yl)-1,2,4-thiadiazolidine-3,5-dione
-
-
2-(2-chloro-4-nitrophenyl)-4-(cyclopropylmethyl)-1,2,4-thiadiazolidine-3,5-dione
-
-
2-(2-chloro-6-methylphenyl)-4-(cyclopropylmethyl)-1,2,4-thiadiazolidine-3,5-dione
-
-
2-(3,5-dimethyl-1,2-oxazol-4-yl)-4-(4-fluorophenyl)-1,2,4-thiadiazolidine-3,5-dione
-
-
2-(3-chloro-4-fluorophenyl)-4-(2,3-dihydro-1H-inden-2-yl)-1,2,4-thiadiazolidine-3,5-dione
-
-
2-(4,6-dimethyl-3-oxo-[1,2]thiazolo[5,4-b]pyridin-2-yl)-N-[2-(4-ethoxyphenyl)ethyl]acetamide
-
-
2-N',2-N',7-N',7-N'-tetramethyl-9H-fluorene-2,7-disulfonohydrazide
-
-
4-(2,3-dihydro-1H-inden-2-yl)-2-[(3-ethylphenyl)methyl]-1,2,4-thiadiazolidine-3,5-dione
-
-
4-(cyclopropylmethyl)-2-(3,5-dimethyl-1,2-oxazol-4-yl)-1,2,4-thiadiazolidine-3,5-dione
-
-
4-(cyclopropylmethyl)-2-[2-(trifluoromethyl)phenyl]-1,2,4-thiadiazolidine-3,5-dione
-
-
4-[4-(propan-2-yl)phenyl]-2-[4-[(trifluoromethyl)sulfanyl]phenyl]-1,2,4-thiadiazolidine-3,5-dione
-
-
5-chloro-N-(3-chloro-4-methoxyphenyl)-2-(methylsulfonyl)pyrimidine-4-carboxamide
-
-
6-O-[3-chloro-4-(6-methoxycarbonylpyridine-2-carbonyl)oxyphenyl] 2-O-methyl pyridine-2,6-dicarboxylate
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-
alafosfalin
-
effective in reducing D-alanine pool levels, alafosfalin forms an external aldimine with the bound PLP cofactor, but is neither racemised nor efficiently hydrolyzed and upon formation of the external aldimine, the phosphonate group interacts with putative catalytic residues and thereby renders them unavailable for catalysis
beta-Chloro-D-alanine
BCDA, the compund is a very poor inhibitor of recombinant Mycobacterium tuberculosis Alr, despite having potent antituberculosis activity, its primary target is glutamate racemase, poor activity oagainst alanine racemase activity, potent antituberculosis activity. BCDA does not inhibit the D-alanine pathway in intact cells, consistent with its poor in vitro activity, it is instead an irreversible mechanism-based inactivator of glutamate racemase (MurI), an upstream enzyme in the same early stage of peptidoglycan biosynthesis. Inhibition kinetics, overview. Glutamate racemase (MurI) is a pyridoxal 5'-phosphate-independent racemase and is therefore unable to undergo the same mechanism of inhibition as Alr with BCDA
additional information
-
N2-substitution of carboxybenzyl-protected derivatives of D,L-cycloserine proceed smoothly with the requisite alkyl halide in the presence of potassium tert-butoxide in dimethylformamide. The synthesised compounds are evaluated for their inhibitory activity against purified Alrs (Alr gene product). Structural modification at the N2 position result in reduced activity in the enzyme assay and underscore the importance of structural modification at N2-position of cycloserine. A compound with CH2CONHOCH3 substituent at N2 position exhibits modest inhibitory activity against purified Alr enzyme from Mycobacterium tuberculosis, Ki = 0.36 mM
-
D-cycloserine
-
DCS, inhibits enzyme Alr irreversibly by covalently bonding to pyridoxal 5'-phosphate, molecular modeling
KM VALUE [mM]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
TURNOVER NUMBER [1/s]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
kcat/KM VALUE [1/mMs-1]
SUBSTRATE
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
Ki VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.086
(4R)-4-amino-3-isoxazolidinone
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.93
2-(4,6-dimethyl-3-oxo-[1,2]thiazolo[5,4-b]pyridin-2-yl)-N-[2-(4-ethoxyphenyl)ethyl]acetamide
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.08
2-phenyl-1-piperidin-1-ylethanethione
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.02
3,3-dihydroxy-1H-quinoline-2,4-dione
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.76
5-chloro-N-(3-chloro-4-methoxyphenyl)-2-(methylsulfonyl)pyrimidine-4-carboxamide
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.68
6-O-[3-chloro-4-(6-methoxycarbonylpyridine-2-carbonyl)oxyphenyl] 2-O-methyl pyridine-2,6-dicarboxylate
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
9
alafosfalin
-
pH and temperature not specified in the publication
0.0086
D-cycloserine
-
pH and temperature not specified in the publication
0.038
ethyl 3-(pyridin-2-ylthio)propanoate
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0086
L-Cycloserine
-
pH and temperature not specified in the publication
0.63
N-benzyl-5-chloro-2-methylsulfonylpyrimidine-4-carboxamide
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
IC50 VALUE [mM]
INHIBITOR
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
IMAGE
0.058
(4R)-4-amino-3-isoxazolidinone
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0049
1,1'-(2-oxido-1,2,5-oxadiazole-3,4-diyl)-bis (1-(2-thienyl))-methanone
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0057
2-(2-hydroxyphenoxy)-N-methylacetamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0077
2-(4,6-dimethyl-3-oxo-[1,2]thiazolo[5,4-b]pyridin-2-yl)-N-[2-(4-ethoxyphenyl)ethyl]acetamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0033
2-(4-methoxyphenyl)-1-morpholin-4-ylethanethione
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0065
2-(4-methylphenyl)-1-morpholin-4-ylethanethione
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0028
2-(hydoxyimino)-6-methyl-2H-benzopyran-3-carboxamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0131
2-(pyridin-3-ylcarbamothioyl sulfanyl)acetic acid
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0016
2-N',2-N',7-N',7-N'-tetramethyl-9H-fluorene-2,7-disulfonohydrazide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.006
2-phenyl-1-piperidin-1-ylethanethione
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0052
3,3-dihydroxy-1H-quinoline-2,4-dione
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0082
5-chloro-N-(3-chloro-4-methoxyphenyl)-2-(methylsulfonyl)pyrimidine-4-carboxamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.001
6-O-[3-chloro-4-(6-methoxycarbonylpyridine-2-carbonyl)oxyphenyl] 2-O-methyl pyridine-2,6-dicarboxylate
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0026
ethyl 3-(pyridin-2-ylthio)propanoate
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.009
N',N',4-trimethylbenzenesulfonohydrazide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0068
N-benzyl-5-chloro-2-methylsulfonylpyrimidine-4-carboxamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0082
N-hydroxy-2-(2-hydroxyphenoxy)acetamide
Mycobacterium tuberculosis
-
in 100 mM Tris-Tricine, pH 8.5, temperature not specified in the publication
0.0264
D-cycloserine
Mycobacterium tuberculosis
-
wild-type enzyme, pH and temperature not specified in the publication
0.712
D-cycloserine
Mycobacterium tuberculosis
-
enzyme mutant R373L, pH and temperature not specified in the publication
1.328
D-cycloserine
Mycobacterium tuberculosis
-
enzyme mutant Y364D, pH and temperature not specified in the publication
pH OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
TEMPERATURE OPTIMUM
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
ORGANISM
COMMENTARY
LITERATURE
UNIPROT
SEQUENCE DB
SOURCE
GENERAL INFORMATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
malfunction
depletion of D-alanine results in rapid loss of viability. The alr mutant is defective for growth in macrophages
physiological function
the enzyme is essential for the organism, it is not possible to generate an alr knockout mutant in the absence of a complementing gene copy or D-alanine in the growth medium
evolution
-
alanine racemase is a fold type III pyridoxal 5'-phosphate-dependent amino acid racemase enzyme
malfunction
-
role of alanine racemase mutations in Mycobacterium tuberculosis D-cycloserine resistance, overview
physiological function
-
alanine racemase plays an essential role in cell wall synthesis as it racemizes L-alanine into D-alanine, a key building block in the biosynthesis of peptidoglycan
additional information
-
amino acid residues 319 and 364 are located directly in the active site. Y364 is involved in the positioning of the phosphate moiety of PLP and thus represents a prominent active site residue in the conserved inner layer of the substrate entrance corridor of Alr
PDB
SCOP
CATH
UNIPROT
ORGANISM
SUBUNIT
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
dimer
formed by two crystallographically different monomers, crystallization data
CRYSTALLIZATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
PROTEIN VARIANTS
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
M319T
-
the M319T mutation is positioned close enough to allow interaction with the D-cycloserine moiety, which, given the large change of the character of the side chain, can strongly affect D-cycloserine reactivity. M319 is located near Y364 and, as a result, it is possible that the M319T mutation alters the interaction with Y364, thereby affecting D-cycloserine inhibition. The M319T mutant enzyme shows minimal inhibition by D-cycloserine, even at 1 mM, the IC50 of this mutant cannot be determined
R373L
-
the mutation is not directly located within the active site but near the dimer interface and close to residues M319 and D320, which play an important role in the makeup of the active site. The replacement of arginine with the short and hydrophobic side chain of leucine might disrupt molecular interactions at the dimer interface as well as destabilize the DCS binding site. The R373L mutation is not located directly within the active site, but also showa a significant increase in resistance to D-cycloserine, with an 27fold increased IC50 compared to the wild-type enzyme
Y364D
-
the mutation to aspartic acid introduces a shorter and negatively charged side chain, which potentially affects pyridoxal 5'-phosphate orientation in the active site. The IC50 of the Y364D mutant for D-cycloserine shows a 50fold increase compared to the wild-type
PURIFICATION (Commentary)
ORGANISM
UNIPROT
LITERATURE
recombinant His-tagged enzyme from Escherichia coli strain BL21(DE3) by nickel affinity chromatography, tag cleavage
CLONED (Commentary)
ORGANISM
UNIPROT
LITERATURE
amplified by PCR, cloned and overexpressed in Escherichia coli BL21, both as native and His-tagged products
expressed in Escherichia coli as an N-terminal polyhistidine fusion
-
gene alr, recombinant His-tagged enzyme in Escherichia coli strain BL21(DE3)
APPLICATION
ORGANISM
UNIPROT
COMMENTARY
LITERATURE
pharmacology
Alr of Mycobacterium tuberculosis is a valid drug target and inhibition of Alr alone results in loss of viability in vitro and in vivo
REF.
AUTHORS
TITLE
JOURNAL
VOL.
PAGES
YEAR
ORGANISM (UNIPROT)
PUBMED ID
SOURCE
Strych, U.; Penland, R.L.; Jimenez, M.; Krause, K.L.; Benedik, M.J.
Characterization of the alanine racemases from two mycobacteria
FEMS Microbiol. Lett.
196
93-98
2001
Mycobacterium avium, Mycobacterium tuberculosis (P9WQA9), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WQA9)
LeMagueres, P.; Im, H.; Ebalunode, J.; Strych, U.; Benedik, M.J.; Briggs, J.M.; Kohn, H.; Krause, K.L.
The 1.9 A crystal structure of alanine racemase from Mycobacterium tuberculosis contains a conserved entryway into the active site
Biochemistry
44
1471-1481
2005
Mycobacterium tuberculosis (P9WQA9), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WQA9)
Priyadarshi, A.; Lee, E.H.; Sung, M.W.; Nam, K.H.; Lee, W.H.; Kim, E.E.; Hwang, K.Y.
Structural insights into the alanine racemase from Enterococcus faecalis
Biochim. Biophys. Acta
1794
1030-1040
2009
Escherichia coli (P0A6B4), Geobacillus stearothermophilus (P10724), Haemophilus influenzae (P45257), Mycobacterium tuberculosis (P9WQA9), Helicobacter pylori (Q1XG01), Enterococcus faecalis v583 (Q837J0), Enterococcus faecalis v583, Pseudomonas aeruginosa (Q9HTQ2), Mycobacterium tuberculosis H37Rv (P9WQA9)
Anthony, K.G.; Strych, U.; Yeung, K.R.; Shoen, C.S.; Perez, O.; Krause, K.L.; Cynamon, M.H.; Aristoff, P.A.; Koski, R.A.
New classes of alanine racemase inhibitors identified by high-throughput screening show antimicrobial activity against Mycobacterium tuberculosis
PLoS ONE
6
e20374
2011
Mycobacterium tuberculosis
Awasthy, D.; Bharath, S.; Subbulakshmi, V.; Sharma, U.
Alanine racemase mutants of Mycobacterium tuberculosis require D-alanine for growth and are defective for survival in macrophages and mice
Microbiology
158
319-327
2012
Mycobacterium tuberculosis (P9WQA9), Mycobacterium tuberculosis, Mycobacterium tuberculosis H37Rv (P9WQA9)
Prosser, G.A.; Rodenburg, A.; Khoury, H.; de Chiara, C.; Howell, S.; Snijders, A.P.; de Carvalho, L.P.
Glutamate racemase is the primary target of beta-chloro-D-alanine in Mycobacterium tuberculosis
Antimicrob. Agents Chemother.
60
6091-6099
2016
Bacillus subtilis (P94556), Mycobacterium tuberculosis (P9WPW9), Mycobacterium tuberculosis, Bacillus subtilis 168 (P94556), Mycobacterium tuberculosis ATCC 25618 / H37Rv (P9WPW9)
Nakatani, Y.; Opel-Reading, H.K.; Merker, M.; Machado, D.; Andres, S.; Kumar, S.S.; Moradigaravand, D.; Coll, F.; Perdigao, J.; Portugal, I.; Schoen, T.; Nair, D.; Devi, K.R.U.; Kohl, T.A.; Beckert, P.; Clark, T.G.; Maphalala, G.; Khumalo, D.; Diel, R.; Klaos, K.; Aung, H.L.; Cook, G.M.; Parkhill, J.; Peaco, S.J.; Swaminathan, S.; Viveiros, M.; Niemann,S.; Krause, K.L.; Kser, C.U.
Role of alanine racemase mutations in Mycobacterium tuberculosis D-cycloserine resistance
Antimicrob. Agents Chemother.
61
e01575-17
2017
Mycobacterium tuberculosis
Azam, M.A.; Jayaram, U.
Inhibitors of alanine racemase enzyme a review
J. Enzyme Inhib. Med. Chem.
31
517-526
2016
Geobacillus stearothermophilus, Bacillus cereus, Chlamydia pneumoniae, Enterobacter sp., Enterococcus faecalis, Lactiplantibacillus plantarum, Lactococcus lactis, Listeria monocytogenes, Methanococcus maripaludis, Staphylococcus aureus, Mycobacterium tuberculosis, Mycolicibacterium smegmatis, no activity in Homo sapiens, Proteus mirabilis, Salmonella enterica subsp. enterica serovar Typhimurium, Serratia marcescens, Erysipelothrix rhusiopathiae, Escherichia coli (P0A6B4), Pseudomonas aeruginosa (Q9HUN4), Pseudomonas aeruginosa ATCC 15692 / DSM 22644 / CIP 104116 / JCM 14847 / LMG 12228 / 1C / PRS 101 / PAO1 (Q9HUN4)
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