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)

X-ray crystallography has been used to examine the binding of three members of the beta-lactam family of antibiotics to the D-alanyl-D-alanine peptidase from Streptomyces R61, a target of penicillins. Cephalosporin C, the monobactam analog of penicillin G and (2,3)-alpha-methylene benzylpenicillin have been mapped at 2.3 A resolution in the form of acyl-enzyme complexes bound to serine 62. On the basis of the positions of these inhibitors, the binding of a tripeptide substrate for the enzyme, L-lysyl-D-alanyl-D-alanine, has been modeled in the active site. The binding of both inhibitors and substrate is facilitated by hydrogen-bonding interactions with a conserved beta-strand (297-303), which is antiparallel to the beta-lactam's acylamide linkage or the substrate's peptide bond. The active site is similar to that in beta-lactamases.
J Mol Biol 1989 Sep 20
PMID:Crystallographic mapping of beta-lactams bound to a D-alanyl-D-alanine peptidase target enzyme. 258 85

Internal deletions close to the C-terminus of the Escherichia coli penicillin binding protein 5 (PBP5, DacA) have defined the C-terminal 18 residues of the protein as essential for membrane binding. This C-terminal sequence is capable of forming a strongly amphiphilic alpha-helix. In this paper we show that the PBP5 amphiphilic helix is able to anchor the periplasmic TEM-beta-lactamase to the inner membrane. In addition, we have demonstrated that mature PBP5 (lacking the N-terminal signal sequence) possesses the ability to bind to the membrane from a soluble form of the protein, showing that translocation across the membrane is unnecessary for anchoring to be established.
Mol Microbiol 1988 Sep
PMID:Analysis of the membrane-binding domain of penicillin-binding protein 5 of Escherichia coli. 305 22

Small (10 residue) C-terminal deletions of PBP5 cause release of this inner membrane protein into the periplasm, indicating disruption of the membrane binding domain. To define the extent of the membrane anchoring domain, oligonucleotide-directed mutagenesis was used to introduce both single amino acid changes and novel restriction sites into the DNA, allowing subsequent construction of precise internal deletions. The 10 C-terminal amino acid residues possess very weak membrane anchoring potential. By extending the sequence to 18 residues membrane binding equivalent to that of authentic PBP5 was achieved. A proline substitution in this region, breaking a potential alpha-helix, also disrupts the membrane binding domain. These results are discussed with respect to the amphiphilicity of the C-terminal sequence when arranged in an alpha-helix.
Mol Microbiol 1987 Jul
PMID:An 18 amino acid amphiphilic helix forms the membrane-anchoring domain of the Escherichia coli penicillin-binding protein 5. 333 Jul 54

The D-alanyl-D-alanine peptidase from Streptomyces sp. R61 is a 37,500 dalton exocellular enzyme that has served as a model for membrane-bound peptidases that are involved in bacterial cell wall biosynthesis. Inhibition of these enzymes by beta-lactam antibiotics ultimately leads to bacterial cell death. The X-ray crystal structure of the R61 D-alanyl-D-alanine peptidase has been solved using multiple isomorphous replacement, simulated annealing and least squares refinement. The space group and unit cell parameters are P2(1)2(1)2(1) with a = 51.1 A, b = 67.3 A and c = 102.4 A. The structure has been refined using 2 sigma data to 1.6 A resolution with a crystallographic R-factor of 0.148. The model contains 347 residues (2938 atoms) and 254 solvent molecules. The overall temperature factor is 9.6 A2, and the estimated coordinate error is 0.14 A. The protein consists of a single polypeptide chain organized into two regions. One region contains a nine-stranded antiparallel beta-sheet with helices on both faces; this region includes both the amino and carboxyl termini. The second region is all helical. Sixty percent of the residues occur in helices or beta-sheet. The reactive Ser62 is found between the two regions of the enzyme at the amino end of the protein's longest-helix which begins with one turn of 3(10) helix and continues with four turns of alpha-helix. The active site is an elongated pocket that contains four basic and four aromatic residues. An oxyanion hole is formed by Ser62 NH and Thr301 NH. The pocket also contains the few key residues that are conserved in all penicillin-binding proteins and beta-lactamases. Two of these residues, Lys65 and Tyr159, are among the 16 side-chains that take on multiple conformations in the R61 crystal structure. Three of the 12 proline rings adopt two conformations which we believe has not been previously reported. There is no anionic acid equivalent to the catalytic Glu166 found in Class A beta-lactamases. Two ordered water molecules (O507 and O644) are found buried in the active site and hydrogen-bonded to each other (2.6 A). O507 could potentially act as the hydrolytic water molecule for deacylation.
J Mol Biol 1995 Nov 24
PMID:The refined crystallographic structure of a DD-peptidase penicillin-target enzyme at 1.6 A resolution. 749 Jul 45

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

The gene bolA has been shown to trigger the formation of osmotically stable round cells when overexpressed in stationary phase. We show that in poor growth conditions bolA is essential for normal cell morphology in stationary phase and under conditions of starvation. During exponential growth bolA promotes round morphology through a mechanism that is strictly dependent on the two main Escherichia colid,d-carboxypeptidases, PBP5 and PBP6. The results show that bolA controls the levels of transcription of dacA (PBP5), dacC (PBP6) and ampC (AmpC), a class C beta-lactamase, thus connecting for the first time penicillin binding proteins (PBPs) and beta-lactamases at the level of gene regulation. Furthermore, PBP5 and PBP6 are shown to be independently regulated and to have distinct effects on the peptidoglycan layer. The evidence presented demonstrates that bolA is a regulator of cell wall biosynthetic enzymes with different roles in cell morphology and cell division.
Mol Microbiol 2002 Sep
PMID:The gene bolA regulates dacA (PBP5), dacC (PBP6) and ampC (AmpC), promoting normal morphology in Escherichia coli. 1235 37


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