title: Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila.
abstract: Kluyvera citrophila penicillin G acylase (KcPGA) has recently attracted increased attention relative to the well studied and commonly used Escherichia coli PGA (EcPGA) because KcPGA is more resilient to harsh conditions and is easier to immobilize for the industrial hydrolysis of natural penicillins to generate the 6-aminopenicillin (6-APA) nucleus, which is the starting material for semi-synthetic antibiotic production. Like other penicillin acylases, KcPGA is synthesized as a single-chain inactive pro-PGA, which upon autocatalytic processing becomes an active heterodimer of α and β chains. Here, the cloning of the pac gene encoding KcPGA and the preparation of a slow-processing mutant precursor are reported. The purification, crystallization and preliminary X-ray analysis of crystals of this precursor protein are described. The protein crystallized in two different space groups, P1, with unit-cell parameters a = 54.0, b = 124.6, c = 135.1 Å, α = 104.1, β = 101.4, γ = 96.5°, and C2, with unit-cell parameters a = 265.1, b = 54.0, c = 249.2 Å, β = 104.4°, using the sitting-drop vapour-diffusion method. Diffraction data were collected at 100 K and the phases were determined using the molecular-replacement method. The initial maps revealed electron density for the spacer peptide.
authors.affiliation: Division of Biochemical Sciences, CSIR - National Chemical Laboratory, Pune 411 008, Maharashtra, India.
authors.forename: Nishant Kumar
authors.initials: NK
authors.lastname: Varshney
authors.forename: Sureshkumar
authors.initials: S
authors.lastname: Ramasamy
authors.forename: James A
authors.initials: JA
authors.lastname: Brannigan
authors.forename: Anthony J
authors.initials: AJ
authors.lastname: Wilkinson
authors.forename: C G
authors.initials: CG
authors.lastname: Suresh
mesh_terms: D001426:Bacterial Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D003001:Cloning, Molecular / Q000379:methods; D003460:Crystallization; D018360:Crystallography, X-Ray; D015964:Gene Expression Regulation, Bacterial; D044086:Kluyvera / Q000201:enzymology / Q000235:genetics*; D009154:Mutation / Q000235:genetics*; D010399:Penicillin Amidase / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017510:Protein Folding
chemical_list: D001426:Bacterial Proteins; D010399:Penicillin Amidase
keywords: Kluyvera citrophila; PGA precursor; autocatalytic processing; pac gene; spacer peptide
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