Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A

Ishino, Fumitoshi; Mitsui, Kazuhiko; Tamaki, Shigeo; Matsuhashi, Michio

doi:10.1016/s0006-291x(80)80166-5

Cited by 129 publications

(88 citation statements)

References 3 publications

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“…This includes members of two opposing groups of enzyme specificities, murein polymerases (synthases) (9) and murein depolymerases (hydrolases) (10). The bifunctional murein transglycosylases/transpeptidases (11,12), known as penicillin-binding proteins (PBPs) 1 1A, 1B and PBP1C, 2 and the transpeptidases PBP2 and PBP3 (13) as well as the endopeptidases PBP4 and PBP7 (14 -16) and the lytic transglycosylases Slt70, MltA, and MltB (17)(18)(19)(20) were found to interact with one another. These findings may reflect an in vivo assembly of murein synthases and murein hydrolases into a multienzyme complex that has been named a "yin yang complex" (3,6).…”

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confidence: 99%

Demonstration of Molecular Interactions between the Murein Polymerase PBP1B, the Lytic Transglycosylase MltA, and the Scaffolding Protein MipA of Escherichia coli

Vollmer¹,

Rechenberg²,

Höltje

1999

Journal of Biological Chemistry

167

177

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Enlargement of the stress-bearing murein sacculus of bacteria depends on the coordinated interaction of murein synthases and hydrolases. To understand the mechanism of interaction of these two classes of proteins affinity chromatography and surface plasmon resonance (SPR) studies were performed. The membranebound lytic transglycosylase MltA when covalently linked to CNBr-activated Sepharose specifically retained the penicillin-binding proteins (PBPs) 1B, 1C, 2, and 3 from a crude Triton X-100 membrane extract of Escherichia coli. In the presence of periplasmic proteins also PBP1A was specifically bound. At least five different non-PBPs showed specificity for MltA-Sepharose. The amino-terminal amino acid sequence of one of these proteins could be obtained, and the corresponding gene was mapped at 40 min on the E. coli genome. This MltAinteracting protein, named MipA, in addition binds to PBP1B, a bifunctional murein transglycosylase/ transpeptidase. SPR studies with PBP1B immobilized to ampicillin-coated sensor chips showed an oligomerization of PBP1B that may indicate a dimerization. Simultaneous application of MipA and MltA onto a PBP1B sensor chip surface resulted in the formation of a trimeric complex. The dissociation constant was determined to be about 10 ؊6 M. The formation of a complex between a murein polymerase (PBP1B) and a murein hydrolase (MltA) in the presence of MipA represents a first step in a reconstitution of the hypothetical murein-synthesizing holoenzyme, postulated to be responsible for controlled growth of the stress-bearing sacculus of E. coli.The cell envelope of Gram-negative bacteria is stabilized by a thin, monolayered exoskeleton consisting of the cross-linked biopolymer murein (peptidoglycan) (1, 3). By forming a bagshaped structure the murein netting, glycan strands crosslinked by peptides, completely encloses the cell. Different models for the growth mechanism of this stress-bearing bacterial exoskeleton, called sacculus, have been put forward (2, 4, 5, 6). Despite the discrepancies in some details, the models all agree on one point, namely that murein synthases and hydrolases have to cooperate with each other to allow for safe insertion of new material into the growing sacculus. Specific protein-protein interactions between a number of enzymes involved in the metabolism of the murein sacculus have been demonstrated by affinity chromatography (7-8). This includes members of two opposing groups of enzyme specificities, murein polymerases (synthases) (9) and murein depolymerases (hydrolases) (10). The bifunctional murein transglycosylases/transpeptidases (11, 12), known as penicillin-binding proteins (PBPs) 1 1A, 1B and PBP1C, 2 and the transpeptidases PBP2 and PBP3 (13) as well as the endopeptidases PBP4 and PBP7 (14 -16) and the lytic transglycosylases Slt70, MltA, were found to interact with one another. These findings may reflect an in vivo assembly of murein synthases and murein hydrolases into a multienzyme complex that has been named a "yin yang complex" (3, 6). Such a murein ...

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confidence: 99%

Demonstration of Molecular Interactions between the Murein Polymerase PBP1B, the Lytic Transglycosylase MltA, and the Scaffolding Protein MipA of Escherichia coli

Vollmer¹,

Rechenberg²,

Höltje

1999

Journal of Biological Chemistry

167

177

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“…The HMW class B PBPs are monofunctional transpeptidases, some of which have essential functions in septation and maintenance of cell shape (8), while the HMW class A PBPs have both transglycosylase and transpeptidase activities (14,42) and appear to be somewhat functionally redundant (17,34). The Bacillus subtilis ponA gene, coding for the HMW class A PBP1, is transcribed predominantly during log-phase growth (34).…”

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confidence: 99%

Penicillin-Binding Protein-Related Factor A Is Required for Proper Chromosome Segregation in Bacillus subtilis

Pedersen

Setlow

2000

J Bacteriol

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Previous work has shown that the ponA gene, encoding penicillin-binding protein 1 (PBP1), is in a two-gene operon with prfA (PBP-related factor A) (also called recU), which encodes a putative 206-residue basic protein (pI ‫؍‬ 10.1) with no significant sequence homology to proteins with known functions. Inactivation of prfA results in cells that grow slower and vary significantly in length relative to wild-type cells. We now show that prfA mutant cells have a defect in chromosome segregation resulting in the production of ϳ0.9 to 3% anucleate cells in prfA cultures grown at 30 or 37°C in rich medium and that the lack of PrfA exacerbates the chromosome segregation defect in smc and spoOJ mutant cells. In addition, overexpression of prfA was found to be toxic for and cause nucleoid condensation in Escherichia coli.Penicillin-binding proteins (PBPs), which catalyze the polymerization and cross-linking of bacterial peptidoglycan, can be divided into three classes based on their amino acid sequence: the low-molecular-weight PBPs and the high-molecular-weight (HMW) class A and class B PBPs (8). The HMW class B PBPs are monofunctional transpeptidases, some of which have essential functions in septation and maintenance of cell shape (8), while the HMW class A PBPs have both transglycosylase and transpeptidase activities (14, 42) and appear to be somewhat functionally redundant (17, 34). The Bacillus subtilis ponA gene, coding for the HMW class A PBP1, is transcribed predominantly during log-phase growth (34). Previous work with B. subtilis mutants lacking one or several of the three known HMW class A PBPs (PBP1, PBP2c, and PBP4) showed that (i) lack of PBP1 results in slower growth, increased cell length, and decreased cell diameter and (ii) PBP1 is functionally more important than PBP2c and PBP4 (34). It was also recently demonstrated that PBP1 localizes to cell division sites and plays an important role in the formation of the peptidoglycan division septum in vegetative cells of B. subtilis (30).The ponA gene is part of a two-gene operon that also includes prfA (PBP-related factor A [note that in the B. subtilis genome database, prfA is called recU]), which is located immediately upstream of and cotranscribed with ponA (33). prfA codes for a putative 206 residue, basic protein (pI of 10.1), which has no significant sequence homology to proteins with known functions. However, DNA sequencing has indicated that genes encoding similar proteins are present in a large number of gram-positive bacteria: a BLAST search (2) of prfA against completed and unfinished microbial genomes (preliminary sequence data were obtained from The Institute of Genomic Research Website at http://www.tigr.org) produced sequences with significant homology from 10 different grampositive organisms, while no prfA homologs were detected in gram-negative bacteria by this analysis. The former organisms include Enterococcus faecalis (54% identity in a 192-aminoacid overlap), Staphylococcus aureus (58% identity in a 167-amino-acid overlap), Streptococcus p...

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“…The average hydropathy of each 19-residue segment of PBP 2 was calculated, and only the NH2-terminal segment (amino acid residues 13 -46) was found to have an average hydropathy value greater than + 1. 6. In this segment there is a long stretch of 25 non-ionic amino acids (residues 21-45, average hydropathy, +2.1).…”

Section: Hydropathy Profirementioning

confidence: 99%

“…These PBPs catalyze the final steps of cell-wall peptidoglycan synthesis and are the killing targets for b-lactam antibiotics. PBP 1A [6], PBP 1B [7 -1 I], and PBP 3 [12] are bifunctional enzymes that catalyze both a peptidoglycan transglycosylase reaction and a penicillin-sensitive peptidoglycan transpeptidase reaction. With PBP 2, however, the two enzymatic activities were demonstrated only in the presence of another protein, RodA [13, 141. The lower-molecular-mass PBPs (PBP 4, PBP 5, and PBP 6) seem to be less important for the growth of E. coli cells, and display penicillin-sensitive DD-peptidase activities such as DD-endopeptidase and D-alanine carboxypeptidase activities 115 -191.…”

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confidence: 99%

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin‐binding protein 2 of Escherichia coli K12

Asoh

Matsuzawa

Ishino

et al. 1986

European Journal of Biochemistry

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We have determined the nucleotide sequence of the pbpA gene encoding penicillin-binding protein (PBP) 2 of Escherichia coli. The coding region for PBP 2 was 1899 base pairs in length and was preceded by a possible promoter sequence and two open reading frames. The primary structure of PBP 2, deduced from the nucleotide sequence, comprised 633 amino acid residues. The relative molecular mass was calculated to be 70867. The deduced sequence agreed with the NH2-terminal sequence of PBP 2 purified from membranes, suggesting that PBP 2 has no signal peptide. The hydropathy profile suggested that the NH,-terminal hydrophobic region (a stretch of 25 non-ionic amino acids) may anchor PBP 2 in the cytoplasmic membrane as an ectoprotein. There were nine homologous segments in the amino acid sequence of PBP 2 when compared with PBP 3 of E. coli. The active-site serine residue of PBP 2 was predicted to be Ser-330. Around this putative active-site serine residue was found the conserved sequence of Ser-Xaa-Xaa-Lys, which has been identified in all of the other E. coli PBPs so far studied (PBPs lA, lB, 3, 5 and 6) and class A and class C B-lactamases. In the higher-molecular-mass PBPs 1 A, lB, 2 and 3, Ser-Xaa-Xaa-Lys-Pro was conserved. In the putative peptidoglycan transpeptidase domain there were six amino acid residues, which are common only in the PBPs of higher molecular mass.In Escherichia coli seven penicillin-binding proteins (PBPs) have been found in the cytoplasmic membrane [l -41. Among them, the higher-molecular-mass PBPs (PBP 1 A, PBP lB, PBP 2, and PBP 3 ) are important for completion of cell cycle of E. coli; PBP 1A and PBP 1 B are responsible for cell elongation [2, 3, 51, PBP 2 for the determination of rod shape of the cell [31,32], 3 [31, 331 and 5 [34] were obtained, and analyses of the peptides confirmed that the predicted active-site sequences were correct. The active-site serine regions of the higher-molecular-mass PBPs display a high sequence homology with each other [31].In this report we present the nucleotide sequence of the pbpA gene encoding PBP 2 of E. coli and the deduced amino acid sequence of this protein. These results prove the homology of the penicillin-binding sites of the higher-molecularmass PBPs of E. coli. Thus, the hypothesis of Tipper and Strominger [35] for the mechanism of the covalent binding of penicillin in all of the essential PBPs of E. coli has been verified.

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Dual enzyme activities of cell wall peptidoglycan synthesis, peptidoglycan transglycosylase and penicillin-sensitive transpeptidase, in purified preparations of Escherichia coli penicillin-binding protein 1A

Cited by 129 publications

References 3 publications

Demonstration of Molecular Interactions between the Murein Polymerase PBP1B, the Lytic Transglycosylase MltA, and the Scaffolding Protein MipA of Escherichia coli

Demonstration of Molecular Interactions between the Murein Polymerase PBP1B, the Lytic Transglycosylase MltA, and the Scaffolding Protein MipA of Escherichia coli

Penicillin-Binding Protein-Related Factor A Is Required for Proper Chromosome Segregation in Bacillus subtilis

Nucleotide sequence of the pbpA gene and characteristics of the deduced amino acid sequence of penicillin‐binding protein 2 of Escherichia coli K12

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