Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Cloning and nucleotide sequencing indicated that transposon Tn1546 from Enterococcus faecium BM4147 encodes a 23,365 Da protein, VanX, required for glycopeptide resistance. The vanX gene was located downstream from genes encoding the VanA ligase and the VanH dehydrogenase which synthesize the depsipeptide D-alanyl-D-lactate (D-Ala-D-Lac). In the presence of ramoplanin, an Enterococcus faecalis JH2-2 derivative producing VanH, VanA and VanX accumulated mainly UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Lac (pentadepsipeptide) and small amounts of UDP-MurNAc-L-Ala-gamma-D-Glu-L-Lys-D-Ala-D-Ala (pentapeptide) in the ratio 49:1. Insertional inactivation of vanX led to increased synthesis of pentapeptide with a resulting change in the ratio of pentadepsipeptide: pentapeptide to less than 1:1. Expression of vanX in E. faecalis and Escherichia coli resulted in production of a D,D-dipeptidase that hydrolysed D-Ala-D-Ala. Pentadepsipeptide, pentapeptide and D-Ala-D-Lac were not substrates for the enzyme. These results establish that VanX is required for production of a D,D-dipeptidase that hydrolyses D-Ala-D-Ala, thereby preventing pentapeptide synthesis and subsequent binding of glycopeptides to D-Ala-D-Ala-containing peptidoglycan precursors at the cell surface.
Mol Microbiol 1994 Sep
PMID:Glycopeptide resistance mediated by enterococcal transposon Tn1546 requires production of VanX for hydrolysis of D-alanyl-D-alanine. 785 21

Vancomycin resistance in Enterococcus faecium requires five genes: vanR, vanS, vanH, vanA, and vanX. The functions and mechanism of four gene products have been known, with VanR/S for signal transduction and transcriptional regulation and VanH/A to synthesize D-Ala-D-lactate. But the function of the fifth gene product, VanX, has been unknown until very recently, when Reynolds and colleagues discovered D-, D-dipeptidase activity in crude extracts of a VanX overproducer [Reynolds, P. E., et al. (1994) Mol. Microbiol. 13, 1065-1070]. We report here the expression of VanX in Escherichia coli and its purification to homogeneity. VanX has been characterized as a metal-activated D-, D-dipeptidase with an optimal pH range of 7-9. The kcat and Km of D-Ala-D-Ala in the absence of divalent metal are determined to be 4.7 s-1 and 1 mM, respectively. However, in the presence of metal cations, kcat can be as high as 788 s-1. VanX is unable to hydrolyze D-Ala-D-lactate, the substituted moiety in the peptidoglycan that leads to vancomycin resistance, not only because of low binding affinity (Ki estimated at 242 mM) but also due to a kcat less than 0.005 s-1. The more than 10(5)-fold differential in catalytic efficiency of VanX for hydrolysis of D-Ala-D-Ala vs D-Ala-D-lactate leaves D-Ala-D-lactate intact for subsequent incorporation into peptidoglycan.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:Overexpression, purification, and characterization of VanX, a D-, D-dipeptidase which is essential for vancomycin resistance in Enterococcus faecium BM4147. 787 24

Transposon Tn1546 from Enterococcus faecium BM4147 mediates high-level resistance to the glycopeptide antibiotics vancomycin and teicoplanin. Tn 1546 encodes a dehydrogenase (VanH) and a ligase (VanA) that synthesize D-alanyl-D-lactate (D-Ala-D-Lac), a D,D-dipeptidase (VanX) that hydrolyses D-Ala-D-Ala and a two-component regulatory system (VanR-VanS) that controls transcription of the vanHAX operon. Strains of Enterococcus faecalis harbouring various copy numbers of the vanRSHAX cluster were tested to determine if there was a correlation between the levels of resistance to glycopeptides, the levels of expression of the corresponding resistance genes and the relative proportions of the different cytoplasmic peptidoglycan precursors. Increased transcription of the vanHAX operon was associated with increased incorporation of D-Ala-D-Lac into peptidoglycan precursors to the detriment of D-Ala-D-Ala, and with a gradual increase in the vancomycin-resistance levels. More complete elimination of D-Ala-D-Ala-containing precursors was required for teicoplanin resistance. The VanY and VanZ proteins also encoded by Tn1546 were not effectors of the regulation of the vanHAX operon but contributed to vancomycin and teicoplanin resistance, respectively. Differences at the regulatory level accounted for phenotypic diversity in acquired glycopeptide resistance by production of D-lac-ending precursors.
Mol Microbiol 1996 Jul
PMID:Quantitative analysis of the metabolism of soluble cytoplasmic peptidoglycan precursors of glycopeptide-resistant enterococci. 884 32

Resistance to glycopeptide antibiotics in enterococci results from the synthesis of peptidoglycan precursors with low affinity for these antibiotics. The resistance proteins are encoded on transposons in VanA and VanB type enterococci and are involved in regulation, synthesis of new resistant precursors and elimination of wild-type sensitive precursors by hydrolysis of D-alanyl-D-alanine (D,D-peptidase activity encoded by vanX) and removal of D-alanine from UDP-N-acetylmuramyl (UDP-MurNAc)-pentapeptide (D,D-carboxypept-idase activity encoded by vanY). The substrate specificities of VanX and VanY ensure that essentially only precursors with low affinity for glycopeptide antibiotics are available for peptidoglycan synthesis in strains induced to resistance.
Cell Mol Life Sci 1998 Apr
PMID:Control of peptidoglycan synthesis in vancomycin-resistant enterococci: D,D-peptidases and D,D-carboxypeptidases. 961 68

VanX is a zinc-dependent D-alanyl-D-alanine dipeptidase that is a critical component in a system that mediates transposon-based vancomycin resistance in enterococci. It is also a key drug target in circumventing clinical vancomycin resistance. The structure of VanX from E. faecium has been solved by X-ray crystallography and reveals a Zn(2+)-dipeptidase with a unique overall fold and a well-defined active site confined within a cavity of limited size. The crystal structures of VanX, the VanX:D-alanyl-D-alanine complex, the VanX:D-alanine complex, and VanX in complex with phosphonate and phosphinate transition-state analog inhibitors, are also presented at high resolution. Structural homology searches of known structures revealed that the fold of VanX is similar to those of two proteins: the N-terminal fragment of murine Sonic hedgehog and the Zn(2+)-dependent N-acyl-D-alanyl-D-alanine carboxypeptidase of S. albus G.
Mol Cell 1998 Jul
PMID:The structure of VanX reveals a novel amino-dipeptidase involved in mediating transposon-based vancomycin resistance. 970 93

Transposon Tn 1546 confers resistance to glycopeptide antibiotics in enterococci and encodes two D,D-peptidases (VanX and VanY) in addition to the enzymes for the synthesis of D-alanyl-D-lactate (D-Ala-D-Lac). VanY was produced in the baculovirus expression system and purified as a proteolytic fragment that lacked the putative N-terminal membrane anchor of the protein. The enzyme was a Zn2+-dependent D,D-carboxypeptidase that cleaved the C-terminal residue of peptidoglycan precursors ending in R-D-Ala-D-Ala or R-D-Ala-D-Lac but not the dipeptide D-Ala-D-Ala. The specificity constants kcat/Km were 17- to 67-fold higher for substrates ending in the R-D-Ala-D-Ala target of glycopeptides. In Enterococcus faecalis, VanY was present in membrane and cytoplasmic fractions, produced UDP-MurNAc-tetrapeptide from cytoplasmic peptidoglycan precursors and was required for high-level glycopeptide resistance in a medium supplemented with D-Ala. The enzyme could not replace the VanX D,D-dipeptidase for the expression of glycopeptide resistance but a G237D substitution in the host D-Ala:D-Ala ligase restored resistance in a vanX null mutant. Deletion of the membrane anchor of VanY led to an active D,D-carboxypeptidase exclusively located in the cytoplasmic fraction that did not contribute to glycopeptide resistance in a D-Ala-containing medium. Thus, VanX and VanY had non-overlapping functions involving the hydrolysis of D-Ala-D-Ala and the removal of D-Ala from membrane-bound lipid intermediates respectively.
Mol Microbiol 1998 Nov
PMID:Requirement of the VanY and VanX D,D-peptidases for glycopeptide resistance in enterococci. 1009 30

VanX and VanY have strict D,D-dipeptidase and D,D-carboxypeptidase activity, respectively, that eliminates production of peptidoglycan precursors ending in D-alanyl-D-alanine (D-Ala-D-Ala) in glycopeptide-resistant enterococci in which the C-terminal D-Ala residue has been replaced by D-lactate. Enterococcus gallinarum BM4174 synthesizes peptidoglycan precursors ending in D-Ala-D-serine (D-Ala-D-Ser) essential for VanC-type vancomycin resistance. Insertional inactivation of the vanC-1 gene encoding the ligase that catalyses synthesis of D-Ala-D-Ser has a polar effect on both D, D-dipeptidase and D,D-carboxypeptidase activities. The open reading frame downstream from vanC-1 encoded a soluble protein designated VanXYC (Mr 22 318), which had both of these activities. It had 39% identity and 74% similarity to VanY in an overlap of 158 amino acids, and contained consensus sequences for binding zinc, stabilizing the binding of substrate and catalysing hydrolysis that are present in both VanX- and VanY-type enzymes. It had very low dipeptidase activity against D-Ala-D-Ser, unlike VanX, and no activity against UDP-MurNAc-pentapeptide[D-Ser], unlike VanY. The introduction of plasmid pAT708(vanC-1,XYC) or pAT717(vanXYC) into vancomycin-susceptible Enterococcus faecalis JH2-2 conferred low-level vancomycin resistance only when D-Ser was present in the growth medium. The peptidoglycan precursor profiles of E. faecalis JH2-2 and JH2-2(pAT708) and JH2-2(pAT717) indicated that the function of VanXYC was hydrolysis of D-Ala-D-Ala and removal of D-Ala from UDP-MurNAc-pentapeptide[D-Ala]. VanC-1 and VanXYC were essential, but not sufficient, for vancomycin resistance.
Mol Microbiol 1999 Oct
PMID:Gene vanXYC encodes D,D -dipeptidase (VanX) and D,D-carboxypeptidase (VanY) activities in vancomycin-resistant Enterococcus gallinarum BM4174. 1056 77

VanX is a zinc-dependent D-Ala-D-Ala amino dipeptidase required for high-level resistance to vancomycin. The enzyme is also able to process dipeptides with bulky C-terminal amino acids [Wu, Z., Wright, G. D., and Walsh, C. T. (1995) Biochemistry 34, 2455-2463]. We took advantage of this observation to design and synthesize the dipeptide-like D-Ala-D-Gly(SPhip-CHF(2))-OH (7) as a potential mechanism-based inhibitor. VanX-mediated peptide cleavage generates a highly reactive 4-thioquinone fluoromethide which is able to covalently react with enzyme nucleophilic residues, resulting in irreversible inhibition. Inhibition of VanX by 7 was time-dependent (K(irr) = 30+/-1 microM; k(inact) = 7.3+/- 0.3 min(-1)) and active site-directed, as deduced from substrate protection experiments. Nucleophilic compounds such as sodium azide, potassium cyanide, and glutathione did not protect the enzyme from inhibition, indicating that the generated nucleophile inactivates VanX before leaving the active site. The failure to reactivate the dead enzyme by gel filtration or pH modification confirmed the covalent nature of the reaction that leads to inactivation. Inactivation was associated with the elimination of fluoride ion as deduced from (19)F NMR spectroscopy analysis and with the production of fluorinated thiophenol dimer 12. These data are consistent with suicide inactivation of VanX by dipeptide 7. The small size of the VanX active site and the presence of a number of nucleophilic side chains at the opening of the active site gorge [Bussiere, D. E., et al. (1998) Mol. Cell 2, 75-84] associated with the high observed partition ratio of 7500+/-500 suggest that the inhibitor is likely to react at the entrance of the active site cavity.
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PMID:Mechanism-based inactivation of VanX, a D-alanyl-D-alanine dipeptidase necessary for vancomycin resistance. 1112 24

The Gram-negative pathogen Salmonella enterica harbours a periplasmic D-Ala-D-Ala dipeptidase (termed PcgL), which confers the ability to grow on D-Ala-D-Ala as sole carbon source. We now demonstrate that inactivation of the pcgL gene renders Salmonella hypervirulent. This phenotype results from the accumulation of peptidoglycan-derived D-Ala-D-Ala in the pcgL mutant and not from an intrinsically faster growth rate. Synthetic D-Ala-D-Ala (but not L-Ala-L-Ala or D-Ala) increased the number of wild-type Salmonella in the liver and spleen of mice within 24 h of injection, suggesting that D-Ala-D-Ala interferes with some aspect of innate immunity. However, the pcgL mutant was unable to grow on D-Ala-D-Ala as sole carbon source and was defective for survival in nutrient-poor conditions. We identified clinical isolates lacking D-Ala-D-Ala dipeptidase activity and unable to grow on D-Ala-D-Ala because of inactivation of the pcgL gene. Our data suggest that genes (such as pcgL) that, when mutated make pathogens more virulent, may be retained because their contribution to pathogen fitness in non-host environments outweighs potential advantages of the hypervirulent vari-ant in the infected host.
Mol Microbiol 2002 Aug
PMID:Conflicting needs for a Salmonella hypervirulence gene in host and non-host environments. 1218 Sep 21

In an effort to structurally probe the metal binding site in VanX, electronic absorption, EPR, and extended x-ray absorption fine structure (EXAFS) spectroscopic studies were conducted on Co(II)-substituted VanX. Electronic spectroscopy revealed the presence of Co(II) ligand field transitions that had molar absorptivities of approximately 100 m(-1) cm(-1), which suggests that Co(II) is five-coordinate in Co(II)-substituted VanX. Low temperature EPR spectra of Co(II)-substituted VanX were simulated using spin Hamiltonian parameters of M(S) = |+/-1/2), E/D = 0.14, g(real(x,y)) = 2.37, and g(real(z)) = 2.03. These parameters lead to the prediction that Co(II) in the enzyme is five-coordinate and that there may be at least one solvent-derived ligand. Single scattering fits of EXAFS data indicate that the metal ions in both native Zn(II)-containing and Co(II)-substituted VanX have the same coordination number and that the metal ions are coordinated by 5 nitrogen/oxygen ligands at approximately 2.0 angstroms. These data demonstrate that Co(II) (and Zn(II) from EXAFS studies) is five-coordinate in VanX in contrast to previous crystallographic studies (Bussiere, D. E., Pratt, S. D., Katz, L., Severin, J. M., Holzman, T., and Park, C. H. (1998) Mol. Cell 2, 75-84). These spectroscopic studies also demonstrate that the metal ion in Co(II)-substituted VanX when complexed with a phosphinate analog of substrate D-Ala-D-Ala is also five-coordinate.
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PMID:A five-coordinate metal center in Co(II)-substituted VanX. 1565 55


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