Functional evolution of the serine β-lactamase active site

Pratt, Robertson

doi:10.1039/b107097p

Cited by 62 publications

(94 citation statements)

References 93 publications

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“…Notably, both D,D-carboxypeptidase activity and ␤-lactam processing proceed through the formation of an acyl-enzyme intermediate followed by deacylation of the enzyme. Based on these data as well as structural and mechanistic information that is available for other PBPs and ␤-lactam-recognizing enzymes (8,9,(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)36), we propose that the acylation mechanism of sPBP3* may occur in four steps. 56 is activated to form the ester bond with the substrate through abstraction of a proton from neighboring Ser 119 by the COOH of the ligand.…”

Section: Discussionmentioning

confidence: 99%

“…It is of interest that all three molecules, which recognize peptidic substrates, harbor a conserved glycine residue at the bottom of the cleft (Gly 161 , Gly 152 , and Gly 144 in sPBP3*, PBP5, and K15, respectively). The importance of the absence of a side chain at this position becomes evident if one considers that it is part of a binding pocket which could accommodate the penultimate (30).…”

Section: Spbp3* Is a Highly Efficient Dd-carboxypeptidase-mentioning

confidence: 99%

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Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Morlot

Pernot

Gouellec

et al. 2005

Journal of Biological Chemistry

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Penicillin-binding proteins (PBPs) are membrane-associated enzymes which perform critical functions in the bacterial cell division process. The single D-Ala,D-Ala (D,D)-carboxypeptidase in Streptococcus pneumoniae, PBP3, has been shown to play a key role in control of availability of the peptidoglycal substrate during cell growth. Here, we have biochemically characterized and solved the crystal structure of a soluble form of PBP3 to 2.8 Å resolution. PBP3 folds into an NH 2 -terminal, D,Dcarboxypeptidase-like domain, and a COOH-terminal, elongated ␤-rich region. The carboxypeptidase domain harbors the classic signature of the penicilloyl serine transferase superfamily, in that it contains a central, five-stranded antiparallel ␤-sheet surrounded by ␣-helices. As in other carboxypeptidases, which are present in species whose peptidoglycan stem peptide has a lysine residue at the third position, PBP3 has a 14-residue insertion at the level of its omega loop, a feature that distinguishes it from carboxypeptidases from bacteria whose peptidoglycan harbors a diaminopimelate moiety at this position. PBP3 performs substrate acylation in a highly efficient manner (k cat /K m ‫؍‬ 50,500 M ؊1 ⅐s ؊1 ), an event that may be linked to its central role in control of pneumococcal peptidoglycan reticulation. A model that places PBP3 poised vertically on the bacterial membrane suggests that its COOH-terminal region could act as a pedestal, placing the active site in proximity to the peptidoglycan and allowing the protein to "skid" on the surface of the membrane, trimming pentapeptides during the cell growth and division processes.Bacterial division is a complex phenomenon that requires the coordination of diverse processes including chromosomal segregation, FtsZ ring-dependent membrane constriction, and cell wall synthesis at the site of septation. The latter process involves the polymerization of glycan chains and transpeptidation of pentapeptidic moieties within the structure of the peptidoglycan, a highly cross-linked mesh that is crucial for maintaining bacterial shape and providing protection from osmotic shock and lysis (1). Both reactions are catalyzed by penicillinbinding proteins (PBPs), 1 membrane-associated molecules, which can be classified as high molecular mass (hmm; often bifunctional) and low molecular mass (lmm; monofunctional) and play key roles in the bacterial life cycle. The pathogenic bacterium Streptococcus pneumoniae offers a unique opportunity for the study of the relationship between cell division and cell wall synthesis, since it carries a relatively simple set of six PBPs, compared with other well studied organisms which present much higher complexity (2). In this organism, PBP1a, -1b, and -2a catalyze both glycosyltransfer and transpeptidation; PBP2b and -2x only catalyze the latter reaction, and PBP3, the single lmm PBP in S. pneumoniae, has been shown to act as a D-Ala,D-Ala (D,D) carboxypeptidase (3).The central role of hmm PBPs in the cell growth and division processes has been recently confirmed ...

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Section: Discussionmentioning

confidence: 99%

Section: Spbp3* Is a Highly Efficient Dd-carboxypeptidase-mentioning

confidence: 99%

Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Morlot

Pernot

Gouellec

et al. 2005

Journal of Biological Chemistry

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“…It is significant that both PBPs and serine-dependent ␤-lactamases share an active site acylation step in their catalytic cycles (29,36). Actually, the active site acylation is believed to have been handed down from the ancestral PBP to the nascent ␤-lactamase (17,22).…”

mentioning

confidence: 99%

“…It has been presumed that in the evolution of PBP from their biosynthetic functions to ␤-lactamases as antibiotic resistance enzymes two key events took place. One was the advent of a hydrolytic deacylation catalytic machinery (22,29). The other was the shedding of the membrane anchor.…”

mentioning

confidence: 99%

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Cytoplasmic-Membrane Anchoring of a Class A β-Lactamase and Its Capacity in Manifesting Antibiotic Resistance

Suvorov

Vakulenko

Mobashery

2007

Antimicrob Agents Chemother

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Bacterial ␤-lactamases are the major causes of resistance to ␤-lactam antibiotics. Three classes of these enzymes are believed to have evolved from ancestral penicillin-binding proteins (PBPs), enzymes responsible for bacterial cell wall biosynthesis. Both ␤-lactamases and PBPs are able to efficiently form acyl-enzyme species with ␤-lactam antibiotics. In contrast to ␤-lactamases, PBPs are unable to efficiently turn over antibiotics and therefore are susceptible to inhibition by ␤-lactam compounds. Although both PBPs and gram-negative ␤-lactamases operate in the periplasm, PBPs are anchored to the cytoplasmic membrane, but ␤-lactamases are not. It is believed that ␤-lactamases shed the membrane anchor in the course of evolution. The significance of this event remains unclear. In an attempt to demonstrate any potential influence of the membrane anchor on the overall biological consequences of ␤-lactamases, we fused the TEM-1 ␤-lactamase to the C-terminal membrane-anchor of penicillin-binding protein 5 (PBP5) of Escherichia coli. The enzyme was shown to express well in E. coli and was anchored to the cytoplasmic membrane. Expression of the anchored enzyme did not result in any changes in antibiotic resistance pattern of bacteria or growth rates. However, in the process of longer coincubation, the organism that harbored the plasmid for the anchored TEM-1 ␤-lactamase lost out to the organism transformed by the plasmid for the nonanchored enzyme over a period of 8 days of continuous growth. The effect would appear to be selection of a variant that eliminates the problematic protein through elimination of the plasmid that encodes it and not structural or catalytic effects at the protein level. It is conceivable that an evolutionary outcome could be the shedding of the sequence for the membrane anchor or alternatively evolution of these enzymes from nonanchored progenitors.

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β‐Lactam Antibiotics

Testero

Fisher

Mobashery

2010

Burger's Medicinal Chemistry and Drug Discovery

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The β‐lactam classes of antibacterials are preeminent in the treatment of bacterial infection due to their unparalleled clinical efficacy and clinical safety. Following the discovery of the penicillins, successive β‐lactam drug discovery has added the cephalosporin, penem cephamycin, clavulanate, monobactam, nocardicin, and carbapenem subclasses. The driving force behind much of this era of discovery is the staggering ability of pathogenic bacteria to adapt previous generations of the β‐lactam by the acquisition and expression of resistance mechanisms. Although many factors contribute to β‐lactam resistance, alterations to the molecular targets of the β‐lactams (the penicillin binding proteins) and the use of enzymes (the β‐lactamases) capable of the hydrolytic deactivation of the β‐lactams are paramount. This review traces the historical development of β‐lactam drug discovery, with emphasis on the most recent progress in the medicinal chemistry, biochemistry, and microbiology of the β‐lactams leading to the discovery of new generation β‐lactam antibacterials effective against the Gram‐negative and ‐positive bacterial pathogens of current medical concern.

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Functional evolution of the serine β-lactamase active site

Cited by 62 publications

References 93 publications

Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Crystal Structure of a Peptidoglycan Synthesis Regulatory Factor (PBP3) from Streptococcus pneumoniae

Cytoplasmic-Membrane Anchoring of a Class A β-Lactamase and Its Capacity in Manifesting Antibiotic Resistance

β‐Lactam Antibiotics

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