Overexpression, purification and biochemical characterization of a class A high-molecular-mass penicillin-binding protein (PBP), PBP1* and its soluble derivative from Mycobacterium tuberculosis (original) (raw)
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Biochemical characterization of the 49 kDa penicillin-binding protein of
The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donorsR"-CONH-CHR#-COX-CHR$-COO− (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor behaving as a simple alterna-tive nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of Mr 52000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, ¯omoxef and cefoxitin.
1996
Cosmid B577, a member of the collection of ordered clones corresponding to the genome of Mycobacterium leprae, contains a gene, provisionally called pon1, that encodes an 821-amino-acid-residue high-molecular-mass class A penicillin-binding protein, provisionally called PBP1. With similar amino acid sequences and modular designs, M. leprae PBP1 is related to Escherichia coli PBP1a and PBP1b, bienzymatic proteins with transglycosylase and transpeptidase activities. When produced in E. coli, His tag-labelled derivatives of M. leprae PBP1 adopt the correct membrane topology, with the bulk of the polypeptide chain on the surface of the plasma membrane. They defy attempts at solubilization with all the detergents tested except cetyltrimethylammonium bromide. The solubilized PBP1 derivatives can be purified by affinity chromatography on Ni 2؉ -nitrilotriacetic acid agarose. They have low affinities for the usual penicillins and cephalosporins.
Biochemical characterization of the 49 kDa penicillin-binding protein of Mycobacterium smegmatis
The Biochemical journal, 1996
The 49 kDa penicillin-binding protein (PBP) of Mycobacterium smegmatis catalyses the hydrolysis of the peptide or S-ester bond of carbonyl donors R1-CONH-CHR2-COX-CHR2-COO- (where X is NH or S). In the presence of a suitable amino acceptor, the reaction partitions between the transpeptidation and hydrolysis pathways, with the amino acceptor, behaving as a simple alternative nucleophile at the level of the acyl-enzyme. By virtue of its N-terminal sequence similarity, the 49 kDa PBP represents one of the class of monofunctional low-molecular-mass PBPs. An immunologically related protein of M(r) 52,000 is present in M. tuberculosis. The 49 kDa PBP is sensitive towards amoxycillin, imipenem, flomoxef and cefoxitin.
Multiple mechanisms of membrane anchoring of Escherichia coli penicillin-binding proteins
FEMS Microbiology Reviews, 1994
The major penicillin-binding proteins (PBPs) of Escherichia coli play vital roles in cell wall biosynthesis and are located in the inner membrane. The high M r PBPs 1A, 1B, 2 and 3 are essential bifunctional transglycosylases/transpeptidases which are thought to be type II integral inner membrane proteins with their C-terminal enzymatic domains projecting into the periplasm. The low M r PBP4 is a DD-carboxypeptidase/endopeptidase, whereas PBPs 5 and 6 are DD-carboxypeptidases. All three low M, PBPs act in the modification of peptidoglycan to allow expansion of the sacculus and are thought to be periplasmic proteins attached with varying affinities to the inner membrane via C-terminal amphiphilic a-helices. It is possible that the PBPs and other inner membrane proteins form a peptidoglycan synthesizing complex to coordinate their activities.
Properties of the Penicillin-Binding Proteins of Escherichia coli K12
European Journal of Biochemistry, 1977
Ben~yl['~C] penicillin binds to six proteins with molecular weights of between 40000 and 91 000 in the inner membrane of Escherichia coli. Two additional binding proteins with molecular weights of 29000 and 32000 were sometimes detected. All proteins were accessible to ben~yl['~C]penicillin in whole cells. Proteins 5 and 6 released bound ben~yI['~C]penicillin with half times of 5 and 19 min at 30 ' C but the other binding proteins showed less than 50% release during a 60-min period at 30 "C. The rate of release of bound penicillin from some of the proteins was greatly stimulated by 2-mei-captoethanol and neutral hydroxylamine. Release of ben~yI['~C]penicillin did not occur ~t ' the binding proteins were denatured in anionic detergent and so was probably enzymic. No additional binding proteins were detected with two ['4C]cephalosporins. These p-lactams bound to either all or some of those proteins to which ben~yl['~C]penicillin bound. No binding proteins have been detected in the outer membrane of E. coli with any p-['4C]lactam. The binding of a range of unlabelled penicillins and cephalosporins were studied by measuring their competition for the binding of berl~yl['~C]penicillin to the six penicillin-binding proteins. These results, together with those obtained by direct binding experiments with P-[14C]lactams, showed that penicillins bind to all six proteins but that at least some cephalosporins fail to bind, or bind very slowly, to proteins 2, 5 and 6, although they bind to the other proteins. Since these cephalosporins inhibited cell division and caused cell lysis at concentrations where we could detect no binding to proteins 2, 5 and 6, we believe that these latter proteins are not the targets at which S-lactams bind to elicit the above physiological responses. The binding properties of proteins 1, 3 and 4 correlate reasonably well with those expected for the above killing targets.
Diversity of Penicillin-binding Proteins
Journal of Biological Chemistry, 2007
Antibiotic-resistant Staphylococcus aureus is a major concern to public health. Methicillin-resistant S. aureus strains are completely resistant to all -lactams antibiotics. One of the main factors involved in methicillin resistance in S. aureus is the penicillin-binding protein, PBP2a. This protein is insensitive to inactivation by -lactam antibiotics such as methicillin. Although other proteins are implicated in high and homogeneous levels of methicillin resistance, the functions of these other proteins remain elusive. Herein, we report for the first time on the putative function of one of these proteins, FmtA. This protein specifically interacts with -lactam antibiotics forming covalently bound complexes. The serine residue present in the sequence motif Ser-X-X-Lys (which is conserved among penicillin-binding proteins and -lactamases) is the active-site nucleophile during the formation of acyl-enzyme species. FmtA has a low binding affinity for -lactams, and it experiences a slow acylation rate, suggesting that this protein is intrinsically resistant to -lactam inactivation. We found that FmtA undergoes conformational changes in presence of -lactams that may be essential to the -lactam resistance mechanism. FmtA binds to peptidoglycan in vitro. Our findings suggest that FmtA is a penicillin-binding protein, and as such, it may compensate for suppressed peptidoglycan biosynthesis under -lactam induced cell wall stress conditions.
Agricultural and biological chemistry, 1985
Penicillin-binding proteins are essential in the cell cycles of bacteria, functioning in the formation of peptidoglycan for lateral growth and cytoplasmic division.1>2) In E. coli, four higher molecular weight PBPs (Mr from 60,000 to 100,000) are essential for the above functions but the importance of lower molecular weight PBPs (49,000 and below) are unknown.2) Mutations causing defects in PBP-5 increased the sensitivity of the cells to /?-lactam antibiotics as the sole discernible phenotype3>4) but the growth in the absence of /Mactams was not inhibited even by complete deletion of its structural gene.5) On the other hand, production of PBP-5 in an excessively large amount was reported to cause rounding of E. coli cells and to be lethal.6) dacAlll917) is a novel mutation; the mutant PBP-5 binds penicillin but can not release it, it has no /?-lactamase activity like the wile-type PBP-5,8>9) and does not show Dalanine carboxypeptidase activity as it can not transfer the bound substrate to water.10) It would be especially interesting to knowthe structures of both the wild-type and mutant-type PBP-5 by amino acid sequencing and X-ray crystallography, as this promises to improve understanding of the mechanism of the D-alanine carboxypeptidase reaction and the relation between the structure and ac
European Journal of Biochemistry, 1978
The penicillin-binding protein that is thought to be the lethal target of penicillin in Bacillus meguterium (protein 1) has been purified to greater than 95 homogeneity. The membrane-bound penicillin-binding proteins were solubilized with a non-ionic detergent and partially separated from each other by ion-exchange chromatography on DEAE-Sepharose CL-6B. Protein 1 was subsequently purified by covalent affinity chromatography on ampicillin-affinose. Bucil1u.y lichmi-,formis contains an equivalent penicillin-binding protein (protein I) that can be more readily purified to virtual homogeneity in a one-step procedure. It was separated from the other penicillin-binding proteins by utilizing the observation that in this organism, this particular protein is the only one whose covalent complex with benzylpenicillin subsequently breaks down. Membranes were treated with saturating concentrations of benzylpenicillin followed by the removal of free penicillin and further incubation to allow the complex between benzylpenicillin and protein 1 to break down. The penicillin-binding proteins were then solubilized and applied to a column of ampicillin-affinose to which only protein 1 was bound as the other penicillin-binding proteins still had benzylpenicillin bound to them. Pure protein 1 was eluted from the affinity resin with hydroxylamine.