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Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila

Varshney, Nishant Kumar and Ramasamy, Sureshkumar and Brannigan, James A. and Wilkinson, Anthony J. and Suresh, C. G. (2013) Cloning, overexpression, crystallization and preliminary X-ray crystallographic analysis of a slow-processing mutant of penicillin G acylase from Kluyvera citrophila. Acta Crystallographica. Section F, Structural Biology and Crystallization Communications, 69 . pp. 925-929. ISSN 1744-3091. https://resolver.caltech.edu/CaltechAUTHORS:20130904-103624532

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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.


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http://dx.doi.org/10.1107/S174430911301943X DOIArticle
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Additional Information:© 2013 International Union of Crystallography. Received 14 May 2013; Accepted 14 July 2013. NKV thanks the Council of Scientific and Industrial Research (CSIR, India) for a research fellowship and the Commonwealth Scholarship Commission, UK and the British Council, UK for a splitsite PhD scholarship. SR thanks the staff at SSRL beamline 12-2 for help with data collection. Operations at SSRL are supported by the US DOE and NIH. The authors thank Ranu Sharma for help in drawing Fig. 1.
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Funding AgencyGrant Number
Council of Scientific and Industrial Research (CSIR) (India)UNSPECIFIED
Commonwealth Scholarship Commission (UK)UNSPECIFIED
British Council (UK)UNSPECIFIED
Department of Energy (DOE)UNSPECIFIED
NIHUNSPECIFIED
Subject Keywords:autocatalytic processing; PGA precursor; spacer peptide; pac gene; Kluyvera citrophila
Record Number:CaltechAUTHORS:20130904-103624532
Persistent URL:https://resolver.caltech.edu/CaltechAUTHORS:20130904-103624532
Usage Policy:No commercial reproduction, distribution, display or performance rights in this work are provided.
ID Code:41081
Collection:CaltechAUTHORS
Deposited By: Tony Diaz
Deposited On:17 Sep 2013 20:16
Last Modified:03 Oct 2019 05:45

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