Penicillin V acylase from Pectobacterium atrosepticum exhibits high specific activity and unique kinetics.
Penicillin V acylases (PVAs, E.C.3.5.11) belong to the Ntn hydrolase super family of enzymes that catalyze the deacylation of the side chain from phenoxymethyl penicillin (penicillin V). Penicillin acylases find use in the pharmaceutical industry for the production of semi-synthetic antibiotics. PVAs employ the N-terminal cysteine residue as catalytic nucleophile and are structurally and evolutionarily related to bile salt hydrolases (BSHs). Here, we report the cloning and characterization of a PVA enzyme from the Gram-negative plant pathogen, Pectobacterium atrosepticum (PaPVA). The enzyme was cloned and expressed in Escherichia coli attaining a very high yield (250 mg/l) and a comparatively high specific activity (430 IU/mg). The enzyme showed marginally better pH and thermo-stability over PVAs characterized from Gram-positive bacteria. The enzyme also showed enhanced activity in presence of organic solvents and detergents. The enzyme kinetics turned out to be significantly different from that of previously reported PVAs, displaying positive cooperativity and substrate inhibition. The presence of bile salts had a modulating effect on PaPVA activity. Sequence analysis and characterization reveal the distinctive nature of these enzymes and underscore the need to study PVAs from Gram-negative bacteria.
Biochemical Sciences Division, National Chemical Laboratory, Dr Homi Bhabha road, Pune - 411008, India.
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VS
Avinash
Biochemical Sciences Division, National Chemical Laboratory, Dr Homi Bhabha road, Pune - 411008, India.
Sureshkumar
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Ramasamy
Biochemical Sciences Division, National Chemical Laboratory, Dr Homi Bhabha road, Pune - 411008, India.
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CG
Suresh
Biochemical Sciences Division, National Chemical Laboratory, Dr Homi Bhabha road, Pune - 411008, India. Electronic address: av.pundle@ncl.res.in.
Archana
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Pundle
D000581:Amidohydrolases / Q000737:chemistry; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D001647:Bile Acids and Salts / Q000737:chemistry; D020134:Catalytic Domain; D003001:Cloning, Molecular; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D044043:Pectobacterium / Q000737:chemistry* / Q000254:growth & development; D010399:Penicillin Amidase / Q000737:chemistry* / Q000235:genetics; D010404:Penicillin V / Q000737:chemistry*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D017422:Sequence Analysis, DNA; D013379:Substrate Specificity; D013696:Temperature
D001426:Bacterial Proteins; D001647:Bile Acids and Salts; D011994:Recombinant Proteins; D000581:Amidohydrolases; D010399:Penicillin Amidase; C023154:choloylglycine hydrolase; D010404:Penicillin V
Cooperative; Enzyme kinetics; Gram-negative; Penicillin V acylase; Substrate inhibition