Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity

Avinash, Vellore Sunder; Panigrahi, Priyabrata; Chand, Deepak; Pundle, Archana; Suresh, C. G.; Ramasamy, Sureshkumar

doi:10.1016/j.jsb.2015.12.008

Cited by 9 publications

(7 citation statements)

References 42 publications

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“…Each ls BSH monomer contains two antiparallel β-sheets sandwiched between α-helices, adopting a four-layered αββα fold of the Ntn hydrolases. Further, the general tetrameric arrangement of monomers is consistent with the physiological assembly described for BSH 12 and other Ntn hydrolase family members 16,17 . Apart from the N-terminus methionine residue, all amino acid residues of ls BSH were resolved in the crystal structure.…”

Section: Resultssupporting

confidence: 80%

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity

Singh

et al. 2019

Sci Rep

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The gut bacterial bile salt hydrolase (BSH) plays a critical role in host lipid metabolism and energy harvest. Therefore, BSH is a promising microbiome target to develop new therapies to regulate obesity in humans and novel non-antibiotic growth promoters for food animals. We previously reported the 1.90 Å apo crystal structure of BSH from Lactobacillus salivarius ( ls BSH). In this study, we soaked the ls BSH crystal with glycocholic acid (GCA), a substrate, and obtained a 2.10 Å structure containing complex of ls BSH bound to GCA and cholic acid (CA), a product. The substrate/product sits in the water-exposed cavity molded by Loops 2 and 3. While the glycine moiety of GCA is exposed into a highly polar pocket, the sterane core of GCA is stabilized by aromatic and hydrophobic interactions. Comparison of product binding with BSH from Clostridium perfringenes reveals a distinct orientation of the sterane core in the binding site. The stability of the substrate- ls BSH complex and the putative catalytic mechanism were explored with molecular dynamics simulations. Site-directed mutagenesis of ls BSH demonstrated that Cys2 and Asn171 are critical for enzymatic activity, while Tyr24, Phe65 and Gln257 contribute to the substrate specificity. Together, this study provides structural insights into BSH-substrate interaction, the mechanism of catalysis and substrate specificity, which facilitate rational design of BSH inhibitors.

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Section: Resultssupporting

confidence: 80%

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity

Singh

et al. 2019

Sci Rep

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“…3 ). A total of 12 motifs were identified, five of which contained previously reported conserved active-site residues ( 17 , 22 ). The sixth active-site residue (corresponding to Cys2) was also determined to be conserved in our alignment ( Fig.…”

Section: Resultsmentioning

confidence: 99%

The Lactobacillus Bile Salt Hydrolase Repertoire Reveals Niche-Specific Adaptation

O’Flaherty

Crawley

Theriot

et al. 2018

mSphere

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Bile acids play an integral role in shaping the gut microbiota and host physiology by regulating metabolic signaling, weight gain, and serum cholesterol and liver triglyceride levels. Given these important roles of bile acids, we investigated the presence of bile salt hydrolase (BSH) in Lactobacillus genomes representing 170 different species, determined strain- and species-specific patterns of occurrences, and expanded on the diversity of the BSH repertoire in this genus. While our data showed that 28% of Lactobacillus species encode BSH proteins, these species are associated mainly with vertebrate-adapted niches, demonstrating selective pressure on lactobacilli to evolve to adapt to specific environments. These new data will allow targeted selection of specific strains of lactobacilli and BSH proteins for future mechanistic studies to explore their therapeutic potential for treating metabolic disorders.

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“…Although the AtPVA tetramer (Fig. 3) possesses a similar non-planar orientation and distance between subunits as PaPVA (Avinash et al 2016a), the angle between the opposite subunits (169.6°) was closer to the planar shape of the PVA from Bacillus sphaericus (171°) than PaPVA (158°). AtPVA shares many similar active site residues with PaPVA including the nucleophilic N-terminal cysteine (C1), and the presence of two Trp residues (W21, W80) in the active site participating in substrate binding.…”

Section: Structural Analysis Of Atpvamentioning

confidence: 94%

“…The lactone ring was housed in the same pocket where the -lactam moiety was bound in the case of Pen V (Avinash et al 2016a) with an Asn residue (N250 in AtPVA or N271 in PaPVA) involved in hydrogen bonding with the NH group of the amide bond. The AHL acyl chain fits into a hydrophobic pocket lined primarily by the two Trp residues in the active site (W23, W87…”

Section: Binding Of Long Chain Ahls To Atpva and Papvamentioning

confidence: 99%

“…In earlier reports, we have characterized the unique biochemical (Avinash et al 2015) and structural (Avinash et al 2016a) features of a highly active PVA from the Gram-negative Pectobacterium atrosepticum (PaPVA). The present study describes the characterization of PVA from another related plant pathogen A. tumefaciens (AtPVA, 62% sequence identity with PaPVA) and elucidates the subtle structural differences between the enzymes.…”

Section: Introductionmentioning

confidence: 99%

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Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa

Sunder

Utari

Ramasamy

et al. 2016

Appl Microbiol Biotechnol

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Virulence pathways in gram-negative pathogenic bacteria are regulated by quorum sensing mechanisms, through the production and sensing of N-acylhomoserine lactone (AHL) signal molecules. Enzymatic degradation of AHLs leading to attenuation of virulence (quorum quenching) could pave the way for the development of new antibacterials. Penicillin V acylases (PVAs) belong to the Ntn hydrolase superfamily, together with AHL acylases. PVAs are exploited widely in the pharmaceutical industry, but their role in the natural physiology of their native microbes is not clearly understood. This report details the characterization of AHL degradation activity by homotetrameric PVAs from two gram-negative plant pathogenic bacteria, Pectobacterium atrosepticum (PaPVA) and Agrobacterium tumefaciens (AtPVA). Both the PVAs exhibited substrate specificity for degrading long-chain AHLs. Exogenous addition of these enzymes into Pseudomonas aeruginosa greatly diminished the production of elastase and pyocyanin and biofilm formation and increased the survival rate in an insect model of acute infection. Subtle structural differences in the PVA active site that regulate specificity for acyl chain length have been characterized, which could reflect the evolution of AHL-degrading acylases in relation to the environment of the bacteria that produce them and also provide strategies for enzyme engineering. The potential for using these enzymes as therapeutic agents in clinical applications and a few ideas about their possible significance in microbial physiology have also been discussed.

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Structural analysis of a penicillin V acylase from Pectobacterium atrosepticum confirms the importance of two Trp residues for activity and specificity

Cited by 9 publications

References 42 publications

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity

The complex structure of bile salt hydrolase from Lactobacillus salivarius reveals the structural basis of substrate specificity

The Lactobacillus Bile Salt Hydrolase Repertoire Reveals Niche-Specific Adaptation

Penicillin V acylases from gram-negative bacteria degrade N-acylhomoserine lactones and attenuate virulence in Pseudomonas aeruginosa

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