Source:http://linkedlifedata.com/resource/pubmed/id/10529248
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| Predicate | Object |
|---|---|
| rdf:type | |
| lifeskim:mentions | |
| pubmed:issue |
42
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| pubmed:dateCreated |
1999-11-24
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| pubmed:abstractText |
Bacteria with either intrinsic or inducible resistance to vancomycin make peptidoglycan (PG) precursors of lowered affinity for the antibiotic by switching the PG-D-Ala-D-Ala termini that are the antibiotic-binding target to either PG-D-Ala-D-lactate or PG-D-Ala-D-Ser as a consequence of altered specificity of the D-Ala-D-X ligases in the cell wall biosynthetic pathway. The VanA ligase of vancomycin-resistant enterococci, a D-Ala-D-lactate depsipeptide ligase, has the ability to recognize and activate the weak nucleophile D-lactate selectively over D-Ala(2) to capture the D-Ala(1)-OPO(3)(2)(-) intermediate in the ligase active site. To ensure this selectivity in catalysis, VanA largely rejects the protonated (NH(3)(+)) form of D-Ala at subsite 2 (K(M2) of 210 mM at pH 7.5) but not at subsite 1. In contrast, the deprotonated (NH(2)) form of D-Ala (K(M2) of 0.66 mM, k(cat) of 550 min(-)(1)) is a 17-fold better substrate compared to D-lactate (K(M) of 0.69 mM, k(cat) of 32 min(-)(1)). The low concentration of the free amine form of D-Ala at physiological conditions (i.e., 0.1% at pH 7.0) explains the inefficiency of VanA in dipeptide synthesis. Mutational analysis revealed a residue in the putative omega-loop region, Arg242, which is partially responsible for electrostatically repelling the protonated form of D-Ala(2). The VanA enzyme represents a subfamily of D-Ala-D-X ligases in which two key active-site residues (Lys215 and Tyr216) in the active-site omega-loop of the Escherichia coli D-Ala-D-Ala ligase are absent. To look for functional complements in VanA, we have mutated 20 residues and evaluated effects on catalytic efficiency for both D-Ala-D-Ala dipeptide and D-Ala-D-lactate depsipeptide ligation. Mutation of Asp232 caused substantial defects in both dipeptide and depsipeptide ligase activity, suggesting a role in maintaining the loop position. In contrast, the H244A mutation caused an increase in K(M2) for D-lactate but not D-Ala, indicating a differential role for His244 in the recognition of the weaker nucleophile D-lactate. Replacement of the VanA omega-loop by that of VanC2, a D-Ala-D-Ser ligase, eliminated D-Ala-D-lactate activity while improving by 3-fold the catalytic efficacy of D-Ala-D-Ala and D-Ala-D-Ser activity.
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| pubmed:grant | |
| pubmed:language |
eng
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| pubmed:journal | |
| pubmed:citationSubset |
IM
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| pubmed:chemical |
http://linkedlifedata.com/resource/pubmed/chemical/3,3-difluoroalanine,
http://linkedlifedata.com/resource/pubmed/chemical/Adenosine Triphosphatases,
http://linkedlifedata.com/resource/pubmed/chemical/Alanine,
http://linkedlifedata.com/resource/pubmed/chemical/Anti-Bacterial Agents,
http://linkedlifedata.com/resource/pubmed/chemical/Arginine,
http://linkedlifedata.com/resource/pubmed/chemical/Bacterial Proteins,
http://linkedlifedata.com/resource/pubmed/chemical/Carbon-Oxygen Ligases,
http://linkedlifedata.com/resource/pubmed/chemical/D-alanylalanine synthetase,
http://linkedlifedata.com/resource/pubmed/chemical/Dipeptides,
http://linkedlifedata.com/resource/pubmed/chemical/Histidine,
http://linkedlifedata.com/resource/pubmed/chemical/Lactic Acid,
http://linkedlifedata.com/resource/pubmed/chemical/Ligands,
http://linkedlifedata.com/resource/pubmed/chemical/Lysine,
http://linkedlifedata.com/resource/pubmed/chemical/Peptide Synthases,
http://linkedlifedata.com/resource/pubmed/chemical/VanA ligase, Bacteria,
http://linkedlifedata.com/resource/pubmed/chemical/VanC protein, Bacteria,
http://linkedlifedata.com/resource/pubmed/chemical/Vancomycin,
http://linkedlifedata.com/resource/pubmed/chemical/acetyl-alanyl-lactate
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| pubmed:status |
MEDLINE
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| pubmed:month |
Oct
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| pubmed:issn |
0006-2960
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| pubmed:author | |
| pubmed:issnType |
Print
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| pubmed:day |
19
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| pubmed:volume |
38
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| pubmed:owner |
NLM
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| pubmed:authorsComplete |
Y
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| pubmed:pagination |
14006-22
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| pubmed:dateRevised |
2007-11-14
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| pubmed:meshHeading |
pubmed-meshheading:10529248-Adenosine Triphosphatases,
pubmed-meshheading:10529248-Alanine,
pubmed-meshheading:10529248-Amino Acid Sequence,
pubmed-meshheading:10529248-Anti-Bacterial Agents,
pubmed-meshheading:10529248-Arginine,
pubmed-meshheading:10529248-Bacterial Proteins,
pubmed-meshheading:10529248-Base Sequence,
pubmed-meshheading:10529248-Carbon-Oxygen Ligases,
pubmed-meshheading:10529248-Dipeptides,
pubmed-meshheading:10529248-Enterococcus,
pubmed-meshheading:10529248-Enzyme Activation,
pubmed-meshheading:10529248-Histidine,
pubmed-meshheading:10529248-Hydrogen-Ion Concentration,
pubmed-meshheading:10529248-Lactic Acid,
pubmed-meshheading:10529248-Leuconostoc,
pubmed-meshheading:10529248-Ligands,
pubmed-meshheading:10529248-Lysine,
pubmed-meshheading:10529248-Molecular Sequence Data,
pubmed-meshheading:10529248-Mutagenesis, Site-Directed,
pubmed-meshheading:10529248-Peptide Synthases,
pubmed-meshheading:10529248-Stereoisomerism,
pubmed-meshheading:10529248-Templates, Genetic,
pubmed-meshheading:10529248-Vancomycin,
pubmed-meshheading:10529248-Vancomycin Resistance
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| pubmed:year |
1999
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| pubmed:articleTitle |
Determinants for differential effects on D-Ala-D-lactate vs D-Ala-D-Ala formation by the VanA ligase from vancomycin-resistant enterococci.
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| pubmed:affiliation |
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115, USA.
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| pubmed:publicationType |
Journal Article,
Comparative Study,
Research Support, U.S. Gov't, P.H.S.
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