Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism 1 1Edited by K. Nagai

McVey, Colin E.; Walsh, Martin A.; Dodson, Guy; Wilson, K.S.; Brannigan, J.A.

doi:10.1006/jmbi.2001.5043

Cited by 156 publications

(147 citation statements)

References 37 publications

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“…Site-directed mutation of these residues may provide clues about the substrate specificity of AHL-acylases. The penicillin acylase family, including penicillin G acylases, cephalosporin acylases, and aculeacin A acylases, is a member of the Ntn-hydrolase superfamily structurally yet has a high degree of substrate specificity (17,26). AiiD from a Ralstonia isolate has also been reported to serve as an AHL-acylase for several reasons, such as no activity on penicillin G and AHLdegrading activity at a nanomolar concentration of AHLs (23).…”

Section: Discussionmentioning

confidence: 99%

Identification of Extracellular N -Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

Park¹,

Kang²,

Jang³

et al. 2005

Appl Environ Microbiol

236

197

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N-Acylhomoserine lactones (AHLs) play an important role in regulating virulence factors in pathogenic bacteria. Recently, the enzymatic inactivation of AHLs, which can be used as antibacterial targets, has been identified in several soil bacteria. In this study, strain M664, identified as a Streptomyces sp., was found to secrete an AHL-degrading enzyme into a culture medium. The ahlM gene for AHL degradation from Streptomyces sp. strain M664 was cloned, expressed heterologously in Streptomyces lividans, and purified. The enzyme was found to be a heterodimeric protein with subunits of approximately 60 kDa and 23 kDa. A comparison of AhlM with known AHL-acylases, Ralstonia strain XJ12B AiiD and Pseudomonas aeruginosa PAO1 PvdQ, revealed 35% and 32% identities in the deduced amino acid sequences, respectively. However, AhlM was most similar to the cyclic lipopeptide acylase from Streptomyces sp. strain FERM BP-5809, exhibiting 93% identity. A mass spectrometry analysis demonstrated that AhlM hydrolyzed the amide bond of AHL, releasing homoserine lactone. AhlM exhibited a higher deacylation activity toward AHLs with long acyl chains rather than short acyl chains. Interestingly, AhlM was also found to be capable of degrading penicillin G by deacylation, showing that AhlM has a broad substrate specificity. The addition of AhlM to the growth medium reduced the accumulation of AHLs and decreased the production of virulence factors, including elastase, total protease, and LasA, in P. aeruginosa. Accordingly, these results suggest that AHL-acylase, AhlM could be effectively applied to the control of AHL-mediated pathogenicity.Many bacterial species employ complex communication systems that link cell density and gene expression to regulate a broad range of biological functions (11,27). Such cell-to-cell communication, termed quorum sensing (QS), depends on the production, diffusion, and recognition of small signal molecules. In gram-negative bacteria, the most intensively studied QS systems rely on the interaction of N-acylhomoserine lactones (AHLs), whose synthesis is directed by LuxI-type AHL synthases, with LuxR-type transcriptional regulators. AHLs share identical homoserine lactone rings yet vary in length and the substitution of an acyl side chain, which determines the signal specificities in different bacterial species. At low population densities, AHLs are present at a basal concentration level. As the population density increases, the accumulated AHLs reach concentrations that allow binding to regulators, and the complexes then activate or inactivate the expression of target genes. In particular, AHL-mediated QS systems play a key role in controlling a range of activities implicated in pathogen and host interactions, such as antibiotic production and the expression of virulence factors (4,5,28,30).Recently, AHLs have been found to be subject to biological inactivation, thereby attracting attention as QS signal interference. Two groups of AHL-degrading enzymes, classified according to the AHL cleavage site, are pro...

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Section: Discussionmentioning

confidence: 99%

Identification of Extracellular N -Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

Park¹,

Kang²,

Jang³

et al. 2005

Appl Environ Microbiol

236

197

View full text Add to dashboard Cite

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“…The structures of the penicillins were either extracted from the PDB if available or modeled from the corresponding SMILES using Corina. As a control, PenG was docked into 1GM7, and the resulting best pose was found to be identical to the conformation obtained experimentally (21). For each docking assay, the distance between the attacking oxygen atom of TthPAC ␤Ser1 and the electrophilic carbon of each of the tested penicillins, as well as the binding energy for this interaction, is detailed in Table 2.…”

Section: Resultsmentioning

confidence: 86%

“…Consequently, in order to mimic the EcoPGA aromatic acyl binding pocket, residues ␣Leu188, ␣Ser189, ␤Leu24, and ␤Ile57 were chosen to be replaced by phenylalanine. Additionally, it has been reported that upon the binding of PenG, EcoPGA undergoes a conformational change that enables the enzyme to accommodate the substrate by moving away the side chain of residues ␣Arg145 and ␣Phe146 (21). In order to enhance binding of PenG to the TthPAC enzyme, ␣Leu188 (the residue structurally aligned with the EcoPGA ␣Arg145) (Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The residues from EcoPGA described here that are involved in the binding of PenG reside on different chains of the enzyme. However, they are structurally linked through a calcium ion that holds the two subunits together and helps to define the structure of the binding pocket (21). From the six calcium-coordinating residues described in EcoPGA (32), TthPAC only conserves three of them (TthPAC ␣Glu195, ␤Asp73, and ␤Asp76), while ␤Glu75 and ␤Gly206 are replacements for ␤Val75 and ␤Pro205, respectively, in EcoPGA.…”

Section: Discussionmentioning

confidence: 99%

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Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

Torres

Cantero

Valle

et al. 2013

Appl Environ Microbiol

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A homologue of the Escherichia coli penicillin acylase is encoded in the genomes of several thermophiles, including in different Thermus thermophilus strains. Although the natural substrate of this enzyme is not known, this acylase shows a marked preference for penicillin K over penicillin G. Three-dimensional models were created in which the catalytic residues and the substrate binding pocket were identified. Through rational redesign, residues were replaced to mimic the aromatic binding site of the E. coli penicillin G acylase. A set of enzyme variants containing between one and four amino acid replacements was generated, with altered catalytic properties in the hydrolyses of penicillins K and G. The introduction of a single phenylalanine residue in position ␣188, ␣189, or ␤24 improved the K m for penicillin G between 9-and 12-fold, and the catalytic efficiency of these variants for penicillin G was improved up to 6.6-fold. Structural models, as well as docking analyses, can predict the positioning of penicillins G and K for catalysis and can demonstrate how binding in a productive pose is compromised when more than one bulky phenylalanine residue is introduced into the active site.

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“…Unfortunately, a clearly defined binding pocket for the b-lactam nucleus cannot be distinguished in the crystal structure of wild-type PGA. However, crystal structures of an inactive PGA mutant in complex with penicillin G (Protein Data Bank [PDB] codes 1FXV [5] and 1GM7 [6]) and of the wild-type PGA in complex with the slowly hydrolyzed penicillin G sulfoxide (PDB code 1GM9 [6]) have been determined and resulted in several residues being implicated in penicillin binding. Mutagenesis of active-site residues resulted in mutant PGAs with an increased propensity to catalyze antibiotic synthesis rather than the hydrolytic side reactions [5,7,8].…”

Section: Enzymes For Sidechain Conversionsmentioning

confidence: 99%

Three-dimensional structures of enzymes useful for ?-lactam antibiotic production

Barends

Yoshida

Dijkstra³

2004

Current Opinion in Biotechnology

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Crystal structures of penicillin acylase enzyme-substrate complexes: structural insights into the catalytic mechanism 1 1Edited by K. Nagai

Cited by 156 publications

References 37 publications

Identification of Extracellular N -Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

Identification of Extracellular N -Acylhomoserine Lactone Acylase from a Streptomyces sp. and Its Application to Quorum Quenching

Engineering the Substrate Specificity of a Thermophilic Penicillin Acylase from Thermus thermophilus

Three-dimensional structures of enzymes useful for ?-lactam antibiotic production

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