Information on EC 6.1.2.1 -

Information on EC 6.1.2.1 - D-alanine-(R)-lactate ligase:

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EC NUMBER	COMMENTARY
6.1.2.1	-

RECOMMENDED NAME	GeneOntology No.
D-alanine-(R)-lactate ligase	-

REACTION	REACTION DIAGRAM	COMMENTARY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
D-alanine + (R)-lactate + ATP = D-alanyl-(R)-lactate + ADP + phosphate		-	-	-	-
D-alanine + (R)-lactate + ATP = D-alanyl-(R)-lactate + ADP + phosphate		Arg301 has a dual function in a sequential reaction mechanism, i.e. substrate orientation in subsite 2 aswell as stabilization of the transition state, overview	Leuconostoc mesenteroides	-	705214

REACTION TYPE	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

PATHWAY	KEGG Link	MetaCyc Link
vancomycin resistance I	-	PWY-6454

SYSTEMATIC NAME	IUBMB Comments
D-alanine:(R)-lactate ligase (ADP-forming)	The product of this enzyme, the depsipeptide D-alanyl-(R)-lactate, can be incorporated into the peptidoglycan pentapeptide instead of the usual D-alanyl-D-alanine dipeptide, which is formed by EC 6.3.2.4, D-alanine---D-alanine ligase. The resulting peptidoglycan does not bind the glycopeptide antibiotics vancomycin and teicoplanin, conferring resistance on the bacteria.

SYNONYMS	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
D-Ala-D-Lac ligase	Bacillus subtilis	-	-	716269
D-alanine-D-lactate ligase	Enterococcus faecium	-	-	706946
D-alanine:D-alanine (D-lactate) ligase (ADP)	Leuconostoc mesenteroides	-	-	705214
D-alanine:D-lactate ligase	Leuconostoc mesenteroides	-	-	705214
depsipeptide ligase	Escherichia coli	-	-	707464
VanA	Enterococcus faecium	-	-	706946
VanA	Bacillus subtilis	-	gene name	716269
VanB	Enterococcus faecium	-	-	707164
VanD	Enterococcus faecium	-	-	707160
VanD4	Enterococcus faecium	-	-	707162
VRSA-9 Ddl	Staphylococcus aureus	-	-	715371

CAS REGISTRY NUMBER	COMMENTARY
No entries in this field

ORGANISM	COMMENTARY	LITERATURE	SEQUENCE CODE	SEQUENCE DB	SOURCE
Bacillus subtilis	gene vanA	716269	-	-
Enterococcus faecalis	V583, isoform VanB	1249	-	-
Enterococcus faecium	-	708102	-	-
Enterococcus faecium	10/96A	707162	-	-
Enterococcus faecium	BM 4660	707164	-	-
Enterococcus faecium	BM4147	707463	-	-
Enterococcus faecium	BM4147, isoform VanA	706946	-	-
Enterococcus faecium	BM4339	707160	-	-
Enterococcus faecium	BM4416	649336	-	-
Enterococcus faecium	isoform VanA	702557	-	-
Enterococcus faecium 10/96A	10/96A	707162	-	-
Enterococcus faecium BM 4660	BM 4660	707164	-	-
Escherichia coli	-	707464	-	-
Leuconostoc mesenteroides	-	705214	-	-
Staphylococcus aureus	-	715371	-	-
Staphylococcus aureus VRSA-9	-	715371	-	-
Thermotoga maritima	-	714541	-	-

GENERAL INFORMATION	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
physiological function	Enterococcus faecium	-	resistance to glycopeptides in Enterococcus faecium BM4416 is due to synthesis of late peptidoglycan precursors ending in D-Ala�D-Lac. Strain BM4416 mainly produces UDP-MurNAc-pentadepsipeptide, 69%, terminating in D-Ala�D-Lac, UDP-MurNAc-tetrapeptide, 24%, and UDP-MurNAc-tripeptide, 7%. No significant amounts of UDP-MurNAc-pentapeptide are found. Constitutive resistance is encoded by a vanD operon closely related to that of Enterococcus faecium BM4339 and also located in the chromosome. Both VanD-type strains produce an inactivated D-Ala:D-Ala ligase due to an insertion in the ddl gene	649336
physiological function	Enterococcus faecium	-	the vanD gene encodes a D-Ala:D-Lac ligase related to VanA and VanB which is not transferable by conjugation. It renders the cells constitutively resistant to vancomycin with a minimum inhibitory concentration MIC of 0.064 mg/ml and to low levels of teicoplanin, MIC 0.004 mg/ml. Cytoplasmic peptidoglycan precursors that accumulate are mainly UDP-MurNAc-pentadepsipeptide, UDP-MurNAc-tetrapeptide, and UDP-MurNAc-tripeptide. The large proportion of UDP-MurNAc-pentadepsipeptide indicates that the mechanism of vancomycin resistance in BM4339 is similar to that in VanA and VanB strains. The presence of UDP-MurNAc-tripeptide implies that the rate of synthesis of D-Ala�D-Ala or D-Ala�D-Lac substrates is limiting	707160
physiological function	Enterococcus faecium	-	Enterococcus faecium 10/96A from Brazil is resistant to vancomycin with a minimum inhibitory concentration MIC of 0.256 mg/ml. Cytoplasmic peptidoglycan precursors from cells of strain 10/96A grown in the presence or absence of 0.004 mg of vancomycin/ml contain in both cases 95% UDP-MurNAc-pentadepsipeptide, 3% UDPMurNAc-pentapeptide, and 2% UDP-MurNAc-tetrapeptide, supporting the role of VanD4 as a D-Ala-D-Lac ligase and indicating that glycopeptide resistance is expressed constitutively	707162
physiological function	Enterococcus faecium	-	in the presence of vancomycin, production of the VanB D-Ala:D-Lac ligase is induced, which overcomes the defect in the synthesis of peptidoglycan precursors ending in D-Ala�D-Ala due to a lack of functional Ddl D-Ala-D-Ala ligase. Vancomycin-dependent strain BM4660 synthesizes mainly UDP-MurNAc-pentadepsipeptide, 44%, whereas large amounts of UDP-MurNActripeptide, 39%, and small amounts of pentapeptide, 12%, and tetrapeptide, 5%, are present. The presence of tripeptide in large quantity suggests that the VanB ligase may not be sufficiently active to synthesize D-Ala�D-Lac as rapidly as tripeptide is produced	707164
physiological function	Enterococcus faecium	-	VanA is a D-alanine:D-alanine ligase of altered substrate specificity. VanA catalyzes ester bond formation between D-alanine and the D-hydroxy acid products of VanH, the best substrate being D-2-hydroxybutyrate. The VanA product D-alanyl-D-2-hydroxybutyrate can then be incorporated into the UDPMurNAc-pentapeptide peptidoglycan precursor	707463
evolution	Staphylococcus aureus	-	the two amino acid substitutions Q260K/A283E via exchange at nucleotide positions 778 and 848, respectively, in VRSA-9 Ddl lead to the ability to synthesize precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. The VRSA-9 Ddl has an altered D-Ala:D-Ala ligase activity relative to that of VRSA-6 with a Km for D-Ala of 2 mM at subsite 1 and 240 mM at subsite 2. The binding affinity for D-Ala at subsite 2 is 14fold lower than that of VRSA-6. The residues at nucleotide positions 778 and 848 are not conserved among D-Ala:DAla ligases and do not interact directly with the substrates. VRSA-9 Ddl shows the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site	715371
additional information	Bacillus subtilis	-	acquisition of vancomycin resistance affects expression of WalRK and PhoPR regulon genes in the phosphate-limited state. Genetic regulation of vancomycin resistance involving also VanA, detailed overview	716269

SUBSTRATE	PRODUCT	REACTION DIAGRAM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY/ Substrate	LITERATURE/ Substrate	COMMENTARY/ Product	LITERATURE/ Product	Reversibility r=reversible ir=irreversible ?=not specified
D-Ala + D-2-hydroxybutanoate + ATP	D-Ala-D-2-hydroxybutanoate + ADP + phosphate		Escherichia coli	-	-	707464	-	-	?
D-Ala + D-2-hydroxybutanoate + ATP	D-Ala-D-2-hydroxybutanoate + ADP + phosphate		Enterococcus faecalis	-	-	1249	-	-	?
D-Ala + D-2-hydroxybutanoate + ATP	D-Ala-D-2-hydroxybutanoate + ADP + phosphate		Enterococcus faecium	-	-	707463	-	-	?
D-Ala + D-2-hydroxyvalerate + ATP	D-Ala-D-2-hydroxyvalerate + ADP + phosphate		Enterococcus faecalis	-	-	1249	-	-	?
D-Ala + D-2-hydroxyvalerate + ATP	D-Ala-D-2-hydroxyvalerate + ADP + phosphate		Enterococcus faecium	-	-	707463	-	-	?
D-Ala + D-Ala + ATP	D-Ala-D-Ala + ADP + phosphate		Escherichia coli	-	-	707464	-	-	?
D-Ala + D-Ala + ATP	D-Ala-D-Ala + ADP + phosphate		Enterococcus faecalis	-	-	1249	-	-	?
D-Ala + D-Ala + ATP	D-Ala-D-Ala + ADP + phosphate		Enterococcus faecium	-	-	707463	-	-	?
D-Ala + D-lactate + ATP	D-Ala-D-lactate + ADP + phosphate		Escherichia coli	-	-	707464	-	-	?
D-Ala + D-lactate + ATP	D-Ala-D-lactate + ADP + phosphate		Enterococcus faecalis	-	-	1249	-	-	?
D-Ala + D-lactate + ATP	D-Ala-D-lactate + ADP + phosphate		Enterococcus faecium	-	-	707463, 708102	-	-	?
D-alanine + (R)-lactate + ATP	D-alanyl-(R)-lactate + ADP + phosphate		Thermotoga maritima	-	mutants variants Y207F, S137G/Y207F, S137F/Y207F, S137T/Y207F, and S137A/Y207F from D-alanine-D-alanine ligase, EC 6.3.2.4. The wild-type D-alanine-D-alanine ligase, EC 6.3.2.4 does not show activity with (R)-lactate	714541	-	-	?
additional information	?	-	Enterococcus faecium	-	reaction proceeds via rapid and reversible formation of the enzyme intermediate D-Ala-phosphate. Reversible cleavage of ATP to ADP and D-Ala-phosphate and reformation of ATP are detectable. D-Ala-phosphate is a common intermediate that serves as the electrophilic substrate in the condensation with D-Lac as nucleophilic cosubstrate	708102	-	-	-
additional information	?	-	Leuconostoc mesenteroides	-	D-alanine:D-alanine (D-lactate) ligase (ADP) from Leuconostoc mesenteroides synthesizes the depsipeptide, D-alanyl-D-lactate, in addition to D-alanyl-D-alanine, when D-alanine and D-lactate are incubated simultaneously. Structure of bound D-alanine and D-lactate at the active subsites and substrate orientations, overview. With D-lactate a bifurcated H-bond from Arg301 to the R-OH of D-lactate may account for its orientation and nucleophile activation. This orientation is observed when the guanidino side chain of this residue is flexible. D-Alanine adopts an orientation that utilizes H-bonding to water 2882 and the D-alanyl phosphate in subsite 1. Both of these orientations provide mechanisms of deprotonation and place the nucleophile within 3.2 A of the electrophilic carbonyl of the D-alanyl phosphate intermediate for formation of the transition state, molecular docking and chiral specificity of lactate and alanine dockings, detailed overview	705214	-	-	-
additional information	?	-	Staphylococcus aureus	-	ligand binding sites of VRSA-9 Ddl, overview	715371	-	-	-
additional information	?	-	Thermotoga maritima	-	substrate specificity of recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, overview	714541	-	-	-

NATURAL SUBSTRATES	NATURAL PRODUCTS	REACTION DIAGRAM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY SUBSTRATE	LITERATURE (Substrate)	COMMENTARY PRODUCT	LITERATURE (Product)
additional information	?	-	Leuconostoc mesenteroides	-	D-alanine:D-alanine (D-lactate) ligase (ADP) from Leuconostoc mesenteroides synthesizes the depsipeptide, D-alanyl-D-lactate, in addition to D-alanyl-D-alanine, when D-alanine and D-lactate are incubated simultaneously. Structure of bound D-alanine and D-lactate at the active subsites and substrate orientations, overview. With D-lactate a bifurcated H-bond from Arg301 to the R-OH of D-lactate may account for its orientation and nucleophile activation. This orientation is observed when the guanidino side chain of this residue is flexible. D-Alanine adopts an orientation that utilizes H-bonding to water 2882 and the D-alanyl phosphate in subsite 1. Both of these orientations provide mechanisms of deprotonation and place the nucleophile within 3.2 A of the electrophilic carbonyl of the D-alanyl phosphate intermediate for formation of the transition state, molecular docking and chiral specificity of lactate and alanine dockings, detailed overview	705214	-	-

COFACTOR	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
ATP	Leuconostoc mesenteroides	-	-	705214	2D-image
ATP	Thermotoga maritima	-	-	714541	2D-image
ATP	Staphylococcus aureus	-	-	715371	2D-image

METALS and IONS	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
Mg2+	Leuconostoc mesenteroides	-	-	705214
Mg2+	Staphylococcus aureus	-	required	715371

INHIBITORS	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate	Enterococcus faecium	-	75% inhibition at 0.5 mM	702557	2D-image
1-[[(4-fluorophenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl phenyl hydrogen phosphate	Enterococcus faecium	-	65% inhibition at 0.5 mM	702557	2D-image
1-[[(4-methoxyphenyl)sulfonyl]amino]-3-(morpholin-4-yl)propan-2-yl dihydrogen phosphate	Enterococcus faecium	-	83% inhibition at 0.5 mM	702557	2D-image
Oxacillin	Staphylococcus aureus	-	VRSA-9 Ddl is less sensitive than VRSA-6 Ddl	715371	2D-image
Vancomycin	Leuconostoc mesenteroides	-	-	705214	2D-image
Vancomycin	Bacillus subtilis	-	-	716269	2D-image

ACTIVATING COMPOUND	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
No entries in this field

KM VALUE [mM]	KM VALUE [mM] Maximum	SUBSTRATE	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
0.6	-	D-2-hydroxybutanoate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
3	-	D-2-hydroxybutanoate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
3.2	-	D-2-hydroxyvalerate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
8.3	-	D-2-hydroxyvalerate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
1.2	-	D-Ala	Enterococcus faecalis	-	cosubstrate D-Ala, binding of N-terminal Ala residue. pH not specified in the publication, temperature not specified in the publication	1249	2D-image
16	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 7.5, 37�C	707464	2D-image
34	-	D-Ala	Enterococcus faecalis	-	cosubstrate D-Ala, binding of C-terminal Ala residue. pH not specified in the publication, temperature not specified in the publication	1249	2D-image
38	-	D-Ala	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
69	-	D-Ala	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	2D-image
80	-	D-Ala	Escherichia coli	-	mutant S150A, pH 7.5, 37�C	707464	2D-image
100	-	D-Ala	Escherichia coli	-	isoform VanA, above 100 mM, pH 7.5, 37�C	707464	2D-image
140	-	D-Ala	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	2D-image
150	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	2D-image
0.88	-	D-lactate	Escherichia coli	-	isoform VanA, pH 7.5, 37�C	707464	2D-image
1.3	-	D-lactate	Escherichia coli	-	isoform VanA, pH 6.0, 37�C	707464	2D-image
1.5	-	D-lactate	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	2D-image
1.6	-	D-lactate	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	2D-image
2.2	-	D-lactate	Escherichia coli	-	mutant S150A, pH 7.5, 37�C	707464	2D-image
5.1	-	D-lactate	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	2D-image
7.1	-	D-lactate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
11.4	-	D-lactate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
27	-	D-lactate	Escherichia coli	-	mutant Y216F, pH 7.5, 37�C	707464	2D-image

TURNOVER NUMBER [1/s]	TURNOVER NUMBER MAXIMUM[1/s]	SUBSTRATE	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
0.25	-	D-2-hydroxybutanoate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
1.8	-	D-2-hydroxybutanoate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
0.42	-	D-2-hydroxyvalerate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
2.6	-	D-2-hydroxyvalerate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
3.3	-	D-Ala	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	2D-image
4.1	-	D-Ala	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
4.92	-	D-Ala	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image
7.5	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	2D-image
10.5	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 7.5, 37�C	707464	2D-image
15	-	D-Ala	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	2D-image
15.7	-	D-Ala	Escherichia coli	-	isoform VanA, pH 7.5, 37�C	707464	2D-image
16.3	-	D-Ala	Escherichia coli	-	mutant S150A, pH 7.5, 37�C	707464	2D-image
0.023	-	D-lactate	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	2D-image
0.057	-	D-lactate	Escherichia coli	-	mutant S150A, pH 7.5, 37�C	707464	2D-image
0.13	-	D-lactate	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	2D-image
0.47	-	D-lactate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	2D-image
0.65	-	D-lactate	Escherichia coli	-	isoform VanA, pH 6.0, 37�C	707464	2D-image
0.67	-	D-lactate	Escherichia coli	-	isoform VanA, pH 7.5, 37�C	707464	2D-image
0.7	-	D-lactate	Escherichia coli	-	mutant Y216F, pH 7.5, 37�C	707464	2D-image
1.17	-	D-lactate	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	2D-image
1.57	-	D-lactate	Enterococcus faecium	-	pH 8.6, 25�C	707463	2D-image

kcat/KM VALUE [1/mMs^-1]	kcat/KM VALUE [1/mMs^-1] Maximum	SUBSTRATE	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
0.084	-	D-2-hydroxybutanoate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	20562
3	-	D-2-hydroxybutanoate	Enterococcus faecium	-	pH 8.6, 25�C	707463	20562
0.05	-	D-2-hydroxyvalerate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	84696
0.81	-	D-2-hydroxyvalerate	Enterococcus faecium	-	pH 8.6, 25�C	707463	84696
0.024	-	D-Ala	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	9036
0.028	-	D-Ala	Escherichia coli	-	isoform VanA, pH 7.5, 37�C	707464	9036
0.05	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	9036
0.12	-	D-Ala	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	9036
0.13	-	D-Ala	Enterococcus faecium	-	pH 8.6, 25�C	707463	9036
0.2	-	D-Ala	Escherichia coli	-	mutant S150A, pH 7.5, 37�C	707464	9036
0.21	-	D-Ala	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	9036
0.65	-	D-Ala	Escherichia coli	-	mutant Y216F, pH 7.5, 37�C	707464	9036
0.014	-	D-lactate	Escherichia coli	-	mutant S150A, pH 6.0, 37�C	707464	9256
0.025	-	D-lactate	Escherichia coli	-	mutant Y216F, pH 6.0, 37�C	707464	9256
0.026	-	D-lactate	Escherichia coli	-	mutant S150A, pH 7.5, 37�C; mutant Y216F, pH 7.5, 37�C	707464	9256
0.0414	-	D-lactate	Enterococcus faecalis	-	pH not specified in the publication, temperature not specified in the publication	1249	9256
0.22	-	D-lactate	Enterococcus faecium	-	pH 8.6, 25�C	707463	9256
0.5	-	D-lactate	Escherichia coli	-	isoform VanA, pH 6.0, 37�C	707464	9256
0.6	-	D-lactate	Escherichia coli	-	isoform VanA, pH 7.5, 37�C	707464	9256
0.74	-	D-lactate	Escherichia coli	-	isoform VanA, pH 8.3, 37�C	707464	9256

Ki VALUE [mM]	Ki VALUE [mM] Maximum	INHIBITOR	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
No entries in this field

IC50 VALUE [mM]	IC50 VALUE [mM] Maximum	INHIBITOR	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	IMAGE
No entries in this field

SPECIFIC ACTIVITY [µmol/min/mg]	SPECIFIC ACTIVITY MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
0.19	-	Thermotoga maritima	-	D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137F/Y207F, pH 7.5, 60�C	714541
0.39	-	Thermotoga maritima	-	D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant Y207F, pH 7.5, 60�C	714541
0.42	-	Thermotoga maritima	-	D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137A/Y207F, pH 7.5, 60�C	714541
1.2	-	Thermotoga maritima	-	D-alanyl-D-lactate depsipeptide formation activity, recombinant D-alanine-D-alanine ligase mutant S137G/Y207F, pH 7.5, 60�C	714541

pH OPTIMUM	pH MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
7.5	-	Thermotoga maritima	-	assay at	714541

pH RANGE	pH RANGE MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

TEMPERATURE OPTIMUM	TEMPERATURE OPTIMUM MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
60	-	Thermotoga maritima	-	assay at	714541

TEMPERATURE RANGE	TEMPERATURE MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

pI VALUE	pI VALUE MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

SOURCE TISSUE	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE	SOURCE
No entries in this field

LOCALIZATION	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	GeneOntology No.	LITERATURE	SOURCE
No entries in this field

PDB	SCOP	CATH	ORGANISM
1e4e, download	SCOP (1e4e)	CATH (1e4e)	Enterococcus faecium

MOLECULAR WEIGHT	MOLECULAR WEIGHT MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
82000	-	Staphylococcus aureus	-	recombinant C-terminally His6-tagged, gel filtration	715371

SUBUNITS	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
?	Enterococcus faecalis	-	x * 38000, SDS-PAGE	1249
dimer	Staphylococcus aureus	-	2 * 41000, recombinant C-terminally His6-tagged, SDS-PAGE	715371

POSTTRANSLATIONAL MODIFICATION	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

Crystallization/COMMENTARY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
VanA crystallizes only in the presence of a phosphinate inhibitor analogue of D-alanine-D-alanine. The crystals diffract to at least 2.5 A resolution and are in the centred orthorhombic space group C2221	Enterococcus faecium	-	706946
VRSA-9 Ddl X-ray diffraction crystal structure determination and analysis	Staphylococcus aureus	-	715371

pH STABILITY	pH STABILITY MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

TEMPERATURE STABILITY	TEMPERATURE STABILITY MAXIMUM	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

GENERAL STABILITY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
No entries in this field

ORGANIC SOLVENT	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
No entries in this field

OXIDATION STABILITY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
No entries in this field

STORAGE STABILITY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
No entries in this field

Purification/COMMENTARY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
recombinant His6-tagged mutant enzymes from Escherichia coli strain BL21(DE3) by nickel affinity chromatography	Thermotoga maritima	-	714541

Cloned/COMMENTARY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
gene vanA, the gene is located on a polycistronic van operon	Bacillus subtilis	-	716269
epxression in Escherichia coli	Enterococcus faecalis	-	1249
expression in Escherichia coli	Enterococcus faecium	-	706946
expression of C-terminally His6-tagged VRSA-9 Ddl	Staphylococcus aureus	-	715371
expression of mutant enzymes in Escherichia coli strain BL21(DE3) as His6-tagged proteins	Thermotoga maritima	-	714541

EXPRESSION	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
No entries in this field

ENGINEERING	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
S150A	Escherichia coli	-	mutant of D-Ala-D-Ala ligase Ddl, mutant has gained depsipeptide ligase activity, i.e. formation of D-Ala-D-Lac, D-Ala-D-hydroxybutyrate, with dipeptide/depsipeptide partition ratios that mimic the pH behaviour of D-Ala-D-lactate ligase VanA. Mutant displays a clear pH-dependent partitioning between the preferred depsipeptide product at low pH and the dipeptide product at high pH	707464
Y216F	Escherichia coli	-	mutant of D-Ala-D-Ala ligase Ddl, mutant has gained depsipeptide ligase activity, i.e. formation of D-Ala-D-Lac, D-Ala-D-hydroxybutyrate, with dipeptide/depsipeptide partition ratios that mimic the pH behaviour of D-Ala-D-lactate ligase VanA. Mutant displays a clear pH-dependent partitioning between the preferred depsipeptide product at low pH and the dipeptide product at high pH	707464
S137A	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
S137A/Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
S137F/Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
S137G/Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
S137T/Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
Y201F/Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
Y207F	Thermotoga maritima	-	site-directed mutagenesis, the mutant D-alanine-D-alanine ligase also shows formation of D-alanyl-D-lactate depsipeptide in contrast to the wild-type enzyme, EC 6.3.2.4, which is inactive with (R)-lactate	714541
additional information	Bacillus subtilis	-	construction of several mutant strains, generating a Bacillus subtilis strain BP341A expressing a depsipeptide-containing (D-Ala-D-Lac) lipid II, phenotype, overview. Strains BP341A-E, that require expression of the VanB-type operon for growth, have mutations in the endogenous Ddl ligase	716269
Q260K/A283E	Staphylococcus aureus	-	the two amino acid substitutions result from point mutations at nucleotide positions 778 and 848, respectively. The mutant enzyme VRSA-9 synthesizes precursors ending in D-Ala-D-Lac (72%) and D-Ala-D-Ala (21%) in the absence of vancomycin. VRSA-9 Ddl shows a 200fold loss of activity and the importance of conformational changes in the dimer interface which can indirectly affect the topology of the active site	715371
additional information	Thermotoga maritima	-	structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for depsipeptide synthesis, overview	714541

Renatured/COMMENTARY	ORGANISM	UNIPROT ACCESSION NO.	LITERATURE
No entries in this field

APPLICATION	ORGANISM	UNIPROT ACCESSION NO.	COMMENTARY	LITERATURE
synthesis	Thermotoga maritima	-	structure-based modification of D-alanine-D-alanine ligase from strain ATCC 43589 for D-alanyl-D-lactate and other depsipeptide synthesis by mutant S137G/Y207F	714541

REF.	AUTHORS	TITLE	JOURNAL	VOL.	PAGES	YEAR	ORGANISM	LINK TO PUBMED	SOURCE
1249	Meziane-Cherif, D.; Badet-Denisot, M.A.; Evers, S.; Courvalin, P.; Badet, B.	Purification and characterization of the VanB ligase associated with type B vancomycin resistance in Enterococcus faecalis V583	FEBS Lett.	354	140-142	1994	Enterococcus faecalis	PubMed
649336	Perichon, B.; Casadewall, B.; Reynolds, P.; Courvalin, P.	Glycopeptide-resistant Enterococcus faecium BM4416 is a VanD-type strain with an impaired D-alanine:D-alanine ligase	Antimicrob. Agents Chemother.	44	1346-1348	2000	Enterococcus faecium	PubMed
702557	Sova, M.; Cadez, G.; Turk, S.; Majce, V.; Polanc, S.; Batson, S.; Lloyd, A.J.; Roper, D.I.; Fishwick, C.W.; Gobec, S.	Design and synthesis of new hydroxyethylamines as inhibitors of D-alanyl-D-lactate ligase (VanA) and D-alanyl-D-alanine ligase (DdlB)	Bioorg. Med. Chem. Lett.	19	1376-1379	2009	Enterococcus faecium	PubMed
705214	Neuhaus, F.C.	Role of Arg301 in substrate orientation and catalysis in subsite 2 of D-alanine:D-alanine (D-lactate) ligase from Leuconostoc mesenteroides: A molecular docking study	J. Mol. Graph. Model.	28	728-734	2010	Leuconostoc mesenteroides	PubMed
706946	Huyton, T.; Roper, D.I.	Crystallization and preliminary X-ray characterization of VanA from Enterococcus faecium BM4147: towards the molecular basis of bacterial resistance to the glycopeptide antibiotic vancomycin	Acta Crystallogr. Sect. D	55	1481-1483	1999	Enterococcus faecium	PubMed
707160	Perichon, B.; Reynolds, P.; Courvalin, P.	VanD-type glycopeptide-resistant Enterococcus faecium BM4339	Antimicrob. Agents Chemother.	41	2016-2018	1997	Enterococcus faecium	PubMed
707162	Dalla Costa, L.M.; Reynolds, P.E.; Souza, H.A.; Souza, D.C.; Palepou, M.F.; Woodford, N.	Characterization of a divergent vanD-type resistance element from the first glycopeptide-resistant strain of Enterococcus faecium isolated in Brazil	Antimicrob. Agents Chemother.	44	3444-3446	2000	Enterococcus faecium	PubMed
707164	San Millan, A.; Depardieu, F.; Godreuil, S.; Courvalin, P.	VanB-type Enterococcus faecium clinical isolate successively inducibly resistant to, dependent on, and constitutively resistant to vancomycin	Antimicrob. Agents Chemother.	53	1974-1982	2009	Enterococcus faecium	PubMed
707463	Bugg, T.D.; Wright, G.D.; Dutka-Malen, S.; Arthur, M.; Courvalin, P.; Walsh, C.T.	Molecular basis for vancomycin resistance in Enterococcus faecium BM4147: biosynthesis of a depsipeptide peptidoglycan precursor by vancomycin resistance proteins VanH and VanA	Biochemistry	30	10408-10415	1991	Enterococcus faecium	PubMed
707464	Park, I.-S.; Lin, C.-H.; Walsh, C.T.	Gain of D-alanyl-D-lactate or D-lactyl-D-alanine synthetase activities in three active-site mutants of the Escherichia coli D-alanyl-D-alanine ligase B	Biochemistry	35	10464-10471	1996	Escherichia coli	PubMed
708102	Healy, V.L.; Mullins, L.S.; Li, X.; Hall, S.E.; Raushel, F.M.; Walsh, C.T.	D-Ala�D-X ligases: evaluation of D-alanyl phosphate intermediate by MIX, PIX and rapid quench studies	Chem. Biol.	7	505-514	2000	Enterococcus faecium	PubMed
714541	Nakagawa, T.; Satake, R.; Sato, M.; Kino, K.	Structure-based modification of D-alanine-D-alanine ligase from Thermotoga maritima ATCC 43589 for depsipeptide synthesis	Biosci. Biotechnol. Biochem.	75	700-704	2011	Thermotoga maritima	PubMed
715371	Meziane-Cherif, D.; Saul, F.A.; Moubareck, C.; Weber, P.; Haouz, A.; Courvalin, P.; Perichon, B.	Molecular basis of vancomycin dependence in VanA-type Staphylococcus aureus VRSA-9	J. Bacteriol.	192	5465-5471	2010	Staphylococcus aureus	PubMed
716269	Bisicchia, P.; Bui, N.K.; Aldridge, C.; Vollmer, W.; Devine, K.M.	Acquisition of VanB-type vancomycin resistance by Bacillus subtilis: the impact on gene expression, cell wall composition and morphology	Mol. Microbiol.	81	157-178	2011	Bacillus subtilis	PubMed

LINKS TO OTHER DATABASES (specific for EC-Number 6.1.2.1)

NCBI: PubMed, Protein, Nucleotide, Structure, Genome, OMIM

IUBMB Enzyme Nomenclature

PROSITE Database of protein families and domains

SYSTERS

Protein Mutant Database

InterPro (database of protein families, domains and functional sites)

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