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Crystal structure of a ?-aminopeptidase from an Australian Burkholderia sp.

ABSTRACT: ?-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal ?-amino acids from synthetic ?-peptides. ?-Peptides can form secondary structures mimicking ?-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of ?-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a ?-aminopeptidase from a Gram-negative Burkholderia sp. that was isolated from activated sludge from a wastewater-treatment plant in Australia, is reported. The crystal structure of BcA5-BapA was determined to a resolution of 2.0?Å and showed a tetrameric assembly typical of the ?-aminopeptidases. Each monomer consists of an ?-subunit (residues 1-238) and a ?-subunit (residues 239-367). Comparison of the structure of BcA5-BapA with those of other known ?-aminopeptidases shows a highly conserved structure and suggests a similar proteolytic mechanism of action.

SUBMITTER: John-White M

PROVIDER: S-EPMC5505242 | biostudies-literature | 2017 Jul

REPOSITORIES: biostudies-literature

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Crystal structure of a β-aminopeptidase from an Australian Burkholderia sp.

John-White Marietta M Dumsday Geoff J GJ Johanesen Priscilla P Lyras Dena D Drinkwater Nyssa N McGowan Sheena S

Acta crystallographica. Section F, Structural biology communications 20170617 Pt 7

β-Aminopeptidases are a unique group of enzymes that have the unusual capability to hydrolyze N-terminal β-amino acids from synthetic β-peptides. β-Peptides can form secondary structures mimicking α-peptide-like structures that are resistant to degradation by most known proteases and peptidases. These characteristics of β-peptides give them great potential as peptidomimetics. Here, the X-ray crystal structure of BcA5-BapA, a β-aminopeptidase from a Gram-negative Burkholderia sp. that was isolate ...[more]

PMID: 28695846

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Project description:BACKGROUND: Leucine aminopeptidase (LAP) has been known to be a housekeeping protease, DNA-binding protein and repressor or activator in the operon regulation of virulence-associated genes in several bacterial species. LAP activity was consistently detected in overnight cultures of Burkholderia pseudomallei, the causative agent of melioidosis and this enzyme was partially purified and characterised in this study. The intra- and inter-species nucleotide and deduced amino acid sequence variation of LAP encoding gene (pepA) was determined. A pepA/PCR-RFLP assay was designed to facilitate the identification of major LAP sequence types amongst clinical and environmental isolates of B. pseudomallei. RESULTS: LAP activity was detected in B. pseudomallei culture supernantants by zymographic analysis. Optimum activity was at pH 9 and stable at 50[degree sign]C. Enhanced enzymatic activity was observed in the presence of metallic ions Mg2+, Ca2+, Na+ and K+. LAP activity was inhibited by EDTA, 1,10-phenanthroline, amastatin, Mn2+ and Zn2+. Sequence analysis of the complete nucleotide and deduced amino acid sequences of LAP-encoding (pepA) gene showed close genetic relatedness to B. mallei (similarity 99.7%/99.6%), but not with B. thailandensis (96.4%/96.4%). Eight pepA sequence types were identified by comparison with a 596 bp DNA fragment encompassing central regions of the pepA gene. A pepA/PCR-RFLP was designed to differentiate pepA sequence types. Based on restriction analysis with StuI and HincII enzymes of the amplified pepA gene, clinical and environmental isolates showed different predominant RFLP types. Type I was the most predominant type amongst 71.4% (65/91) of the clinical isolates, while Type II was predominant in 55.6% (5/9) of the environmental isolates. CONCLUSIONS: This study showed that LAP is a secretory product of B. pseudomallei with features similar to LAP of other organisms. Identification of major LAP sequence types of B. pseudomallei was made possible based on RFLP analysis of the pepA gene. The high LAP activity detected in both B. pseudomallei and B. thailandensis, suggests that LAP is probably a housekeeping enzyme rather than a virulence determinant.

Dataset Information

Crystal structure of a ?-aminopeptidase from an Australian Burkholderia sp.

Publications

Crystal structure of a β-aminopeptidase from an Australian Burkholderia sp.

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