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Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

ABSTRACT: The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid sequence of PBP 2a from strain 27r revealed a hydrophobic region at the amino terminus that possessed characteristics of an uncleaved signal peptide such as those found in type II integral membrane proteins. Site-specific mutagenesis was used to modify the strain 27r mecA gene to permit removal of the region encoding the putative transmembrane region (amino acids 2 to 22). When it was expressed in E. coli, the modified mecA gene from strain 27r encoded a water-soluble form of PBP 2a that was detectable in the cytoplasm of transformants. The water-soluble form of PBP 2a protein from S. aureus 27r retained the same binding efficiency for beta-lactam antibiotics as the unmodified membrane-bound PBP 2a from S. aureus 27r.

SUBMITTER: Wu CY

PROVIDER: S-EPMC190552 | biostudies-other | 1992 Mar

REPOSITORIES: biostudies-other

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Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

Wu C Y CY Hoskins J J Blaszczak L C LC Preston D A DA Skatrud P L PL

Antimicrobial agents and chemotherapy 19920301 3

The mecA gene from methicillin-resistant Staphylococcus aureus 27r, which encodes the membrane-bound penicillin-binding protein 2a (PBP 2a), was cloned, sequenced, and expressed in Escherichia coli. PBP 2a is the major factor that mediates methicillin resistance in staphylococci. The DNA sequence of the mecA gene from strain 27r was greater than 99% identical to the DNA sequence of other S. aureus mecA genes and the mecA gene from Staphylococcus epidermidis. Analysis of the deduced amino acid se ...[more]

PMID: 1622161

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Project description:The mechanism of the ?-lactam antibacterials is the functionally irreversible acylation of the enzymes that catalyze the cross-linking steps in the biosynthesis of their peptidoglycan cell wall. The Gram-positive pathogen Staphylococcus aureus uses one primary resistance mechanism. An enzyme, called penicillin-binding protein 2a (PBP2a), is brought into this biosynthetic pathway to complete the cross-linking. PBP2a effectively discriminates against the ?-lactam antibiotics as potential inhibitors, and in favor of the peptidoglycan substrate. The basis for this discrimination is an allosteric site, distal from the active site, that when properly occupied concomitantly opens the gatekeeper residues within the active site and realigns the conformation of key residues to permit catalysis. We address the molecular basis of this regulation using crystallographic studies augmented by computational analyses. The crystal structures of three ?-lactams (oxacillin, cefepime, ceftazidime) complexes with PBP2a-each with the ?-lactam in the allosteric site-defined (with preceding PBP2a structures) as the "open" or "partially open" PBP2a states. A particular loop motion adjacent to the active site is identified as the driving force for the active-site conformational change that accompanies active-site opening. Correlation of this loop motion to effector binding at the allosteric site, in order to identify the signaling pathway, was accomplished computationally in reference to the known "closed" apo-PBP2a X-ray crystal structure state. This correlation enabled the computational simulation of the structures coinciding with initial peptidoglycan substrate binding to PBP2a, acyl enzyme formation, and acyl transfer to a second peptidoglycan substrate to attain cross-linking. These studies offer important insights into the structural bases for allosteric site-to-active site communication and for ?-lactam mimicry of the peptidoglycan substrates, as foundational to the mechanistic understanding of emerging PBP2a resistance mutations.

Dataset Information

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

Publications

Construction of a water-soluble form of penicillin-binding protein 2a from a methicillin-resistant Staphylococcus aureus isolate.

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