Mutational analysis of active-site residues of the enterococcal D-ala-D-Ala dipeptidase VanX and comparison with Escherichia coli D-ala-D-Ala ligase and D-ala-D-Ala carboxypeptidase VanY.
(1/335)
BACKGROUND: Vancomycin-resistant enterococci are pathogenic bacteria that attenuate antibiotic sensitivity by producing peptidoglycan precursors that terminate in D-Ala-D-lactate rather than D-Ala-D-Ala. A key enzyme in effecting antibiotic resistance is the metallodipeptidase VanX, which reduces the cellular pool of the D-Ala-D-Ala dipeptide. RESULTS: We constructed eleven mutants, using the recently determined VanX structure as a basis, to investigate residue function. Mutating Asp142 or Ser114 showed a large effect principally on KM, consistent with roles in recognition of the D-Ala-D-Ala termini. The drastic reduction or absence of activity in the Arg71 mutants correlates with a role in the stabilization of an anionic tetrahedral transition state. Three residues of the Escherichia coli D-Ala-D-Ala ligase (Ddl), Glu15, Ser 281 and Arg255, are similarly conserved and have equivalent functions with respect to VanX, consistent with a convergent evolution of active sites to bind D-Ala-D-Ala and lower energy barriers for formation of the tetrahedral intermediate and transition states. In the N-acyl-D-Ala-D-Ala carboxypeptidase VanY, all active-site residues are conserved (except for the two responsible for recognition of the dipeptide amino terminus). CONCLUSIONS: The mutagenesis results support structure-based functional predictions and explain why the VanX dipeptidase and Ddl ligase show narrow specificity for the D,D-dipeptide substrate. The results reveal that VanX and Ddl, two enzymes that use the same substrate but proceed in opposite directions driven by distinct cofactors (zinc versus ATP), evolved similar architectural solutions to substrate recognition and catalysis acceleration. VanY sequence analysis predicts an active site and mechanism of reaction similar to VanX. (+info)
Characterization of a prolidase from Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 with an unusual regulation of biosynthesis.
(2/335)
Lactobacillus delbrueckii subsp. bulgaricus CNRZ 397 (Lb. bulgaricus) is characterized by a high level of peptidase activities specific to proline-containing peptides. A prolidase (PepQ, EC 3.4.13.9) was purified to homogeneity and characterized as a strict dipeptidase active on X-Pro dipeptides, except Gly-Pro and Pro-Pro. The values for Km and Vmax were, respectively, 2.2 mM and 0.33 mmol min(-1) mg(-1), with Leu-Pro as the substrate. The enzyme exhibited optimal activity at 50 degrees C and pH 6.0, and required the presence of Zn2+. Size exclusion chromatographies and SDS-PAGE analysis led to the conclusion that this prolidase was a homodimer. Antibodies raised against the purified protein allowed the detection of PepQ among several Lactobacillus species but not lactococci. The pepQ gene and the upstream region were isolated and sequenced. The deduced peptide sequence showed that PepQ belongs to the M24 family of metallopeptidases. The pepR1 gene is located immediately upstream of pepQ and its product is homologous to the transcription factor CcpA, which is involved in catabolite repression of catabolic operons from Gram-positive bacteria. The pepR1-pepQ intergenic region contains a consensus catabolite-responsive element (CRE) which could be a target for PepR1 protein. Moreover, in contrast to other proline-specific enzymes from Lb. bulgaricus, PepQ biosynthesis was shown to be dependent on the composition of the culture medium, but not on the peptide concentration. A possible regulation mechanism is discussed. (+info)
Similarities between a dipeptide hydrolase from brush-border and cytosol fractions of guinea-pig intestinal mucosa.
(3/335)
A dipeptide hydrolase from the brush border of guinea-pig intestinal mucosa was purified. The enzyme resembles another dipeptide hydrolase isolated from the cytosol fraction of intestinal mucosa. Studies on the binding of cytosol peptide hydrolase to brush-border membranes indicate that the enzyme found in the brush border may be a cytoplasmic contaminant. (+info)
A periplasmic D-alanyl-D-alanine dipeptidase in the gram-negative bacterium Salmonella enterica.
(4/335)
The VanX protein is a D-alanyl-D-alanine (D-Ala-D-Ala) dipeptidase essential for resistance to the glycopeptide antibiotic vancomycin. While this enzymatic activity has been typically associated with vancomycin- and teicoplainin-resistant enterococci, we now report the identification of a D-Ala-D-Ala dipeptidase in the gram-negative species Salmonella enterica. The Salmonella enzyme is only 36% identical to VanX but exhibits a similar substrate specificity: it hydrolyzes D-Ala-D-Ala, DL-Ala-DL-Phe, and D-Ala-Gly but not the tripeptides D-Ala-D-Ala-D-Ala and DL-Ala-DL-Lys-Gly or the dipeptides L-Ala-L-Ala, N-acetyl-D-Ala-D-Ala, and L-Leu-Pro. The Salmonella dipeptidase gene, designated pcgL, appears to have been acquired by horizontal gene transfer because pcgL-hybridizing sequences were not detected in related bacterial species and the G+C content of the pcgL-containing region (41%) is much lower than the overall G+C content of the Salmonella chromosome (52%). In contrast to wild-type Salmonella, a pcgL mutant was unable to use D-Ala-D-Ala as a sole carbon source. The pcgL gene conferred D-Ala-D-Ala dipeptidase activity upon Escherichia coli K-12 but did not allow growth on D-Ala-D-Ala. The PcgL protein localizes to the periplasmic space of Salmonella, suggesting that this dipeptidase participates in peptidoglycan metabolism. (+info)
Cobalt proteins.
(5/335)
In the form of vitamin B12, cobalt plays a number of crucial roles in many biological functions. However, recent studies have provided information on the biochemistry and bioinorganic chemistry of several proteins containing cobalt in a form other than that in the corrin ring of vitamin B12. To date, eight noncorrin-cobalt-containing enzymes (methionine aminopeptidase, prolidase, nitrile hydratase, glucose isomerase, methylmalonyl-CoA carboxytransferase, aldehyde decarbonylase, lysine-2,3-aminomutase, and bromoperoxidase) have been isolated and characterized. A cobalt transporter is involved in the metallocenter biosynthesis of the host cobalt-containing enzyme, nitrile hydratase. Understanding the differences between cobalt and nickel transporters might lead to drug development for gastritis and peptic ulceration. (+info)
Membrane dipeptidase is the receptor for a lung-targeting peptide identified by in vivo phage display.
(6/335)
In vivo phage display is a powerful method to study organ- and tissue-specific vascular addresses. Using this approach, peptides capable of tissue-specific homing can be identified by performing a selection for that trait in vivo. We recently showed that the CGFECVRQCPERC (termed GFE-1) peptide can selectively bind to mouse lung vasculature after an intravenous injection. Our aim in the present study was to identify the receptor for this lung-homing peptide. By using affinity chromatography, we isolated a 55-kDa lung cell-surface protein that selectively binds to the GFE-1 peptide. Protein sequencing established the identity of the receptor as membrane dipeptidase (MDP), a cell-surface zinc metalloprotease involved in the metabolism of glutathione, leukotriene D4, and certain beta-lactam antibiotics. Phage particles displaying the GFE-1 peptide selectively bind to COS-1 cells transfected with the murine MDP cDNA. Moreover, the synthetic GFE-1 peptide could inhibit MDP activity. By establishing MDP as the receptor for the GFE-1 peptide, our results suggest potential applications for both MDP and the GFE-1 peptide in delivery of compounds to the lungs. This work also demonstrates that cell-surface proteases can be involved in tissue-specific homing. (+info)
Pharmacokinetic changes of a new carbapenem, DA-1131, after intravenous administration to spontaneously hypertensive rats and deoxycorticosterone acetate-salt-induced hypertensive rats.
(7/335)
The pharmacokinetics of a new carbapenem, DA-1131, were compared after i.v. administration of the drug, 50 mg/kg, to spontaneously hypertensive rats (SHRs) at 16 weeks of age (an animal model for human primary hypertension) and at 6 weeks of age (corresponding to the early phase of the development of hypertension, at which time blood pressure remains within the normotensive range) and their respective age-matched control normotensive Kyoto-Wistar rats (KW rats), and deoxycorticosterone acetate-salt-induced hypertensive rats at 16 weeks of age (an animal model for human secondary hypertension) and their age-matched control Sprague-Dawley rats. The total area under the plasma concentration-time curve from time zero to time infinity (AUC) (4720 versus 7070 microg x min/ml) was significantly smaller, and the nonrenal clearance (CLNR) (5.37 versus 3.57 ml/min/kg) was significantly faster in 16-week-old SHRs than those in their control KW rats. Similar results were also obtained from 6-week-old SHRs in AUC (3800 versus 4680 microg x min/ml) and CLNR (7.73 versus 3.31 ml/min/kg). However, the values were reversed in 16-week-old deoxycorticosterone acetate-salt rats in AUC (5310 versus 3870 microg.min/ml) and CLNR (2.57 versus 4.90 ml/min/kg). The significantly faster CLNR of DA-1131 in both 6- and 16-week-old SHRs could be supported at least partly by the results of the in vitro metabolism with kidney homogenate and considerably greater total renal dehydropeptidase-I activity. The data above indicated that the significantly faster CLNR of DA-1131 in 16-week-old SHRs than that in their age-matched control KW rats was due to any hereditary characteristics of SHRs and was not due to the hypertensive state itself. (+info)
Escherichia coli mutants lacking all possible combinations of eight penicillin binding proteins: viability, characteristics, and implications for peptidoglycan synthesis.
(8/335)
The penicillin binding proteins (PBPs) synthesize and remodel peptidoglycan, the structural component of the bacterial cell wall. Much is known about the biochemistry of these proteins, but little is known about their biological roles. To better understand the contributions these proteins make to the physiology of Escherichia coli, we constructed 192 mutants from which eight PBP genes were deleted in every possible combination. The genes encoding PBPs 1a, 1b, 4, 5, 6, and 7, AmpC, and AmpH were cloned, and from each gene an internal coding sequence was removed and replaced with a kanamycin resistance cassette flanked by two res sites from plasmid RP4. Deletion of individual genes was accomplished by transferring each interrupted gene onto the chromosome of E. coli via lambda phage transduction and selecting for kanamycin-resistant recombinants. Afterwards, the kanamycin resistance cassette was removed from each mutant strain by supplying ParA resolvase in trans, yielding a strain in which a long segment of the original PBP gene was deleted and replaced by an 8-bp res site. These kanamycin-sensitive mutants were used as recipients in further rounds of replacement mutagenesis, resulting in a set of strains lacking from one to seven PBPs. In addition, the dacD gene was deleted from two septuple mutants, creating strains lacking eight genes. The only deletion combinations not produced were those lacking both PBPs 1a and 1b because such a combination is lethal. Surprisingly, all other deletion mutants were viable even though, at the extreme, 8 of the 12 known PBPs had been eliminated. Furthermore, when both PBPs 2 and 3 were inactivated by the beta-lactams mecillinam and aztreonam, respectively, several mutants did not lyse but continued to grow as enlarged spheres, so that one mutant synthesized osmotically resistant peptidoglycan when only 2 of 12 PBPs (PBPs 1b and 1c) remained active. These results have important implications for current models of peptidoglycan biosynthesis, for understanding the evolution of the bacterial sacculus, and for interpreting results derived by mutating unknown open reading frames in genome projects. In addition, members of the set of PBP mutants will provide excellent starting points for answering fundamental questions about other aspects of cell wall metabolism. (+info)