The Open Microbiology Journal




ISSN: 1874-2858 ― Volume 13, 2019

Biochemical and Genetic Characterization of PspE and GlpE, Two Single-domain Sulfurtransferases of Escherichia coli



Hui Cheng, Janet L Donahue, Scott E Battle, W. Keith Ray, Timothy J Larson*
Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 USA

Abstract

The pspE and glpE genes of Escherichia coli encode periplasmic and cytoplasmic single-domain rhodaneses, respectively, that catalyzes sulfur transfer from thiosulfate to thiophilic acceptors. Strains deficient in either or both genes were constructed. Comparison of rhodanese activity in these strains revealed that PspE provides 85% of total rhodanese activity, with GlpE contributing most of the remainder. PspE activity was four times higher during growth on glycerol versus glucose, and was not induced by conditions that induce expression of the psp regulon. The glpE/pspE mutants displayed no apparent growth phenotypes, indicating that neither gene is required for biosynthesis of essential sulfur-containing molecules. PspE was purified by using cation exchange chromatography. Two distinct active peaks were eluted and differed in the degree of stable covalent modification, as assessed by mass spectrometry. The peak eluting earliest contained the equivalent mass of two additional sulfur atoms, whereas the second peak contained mainly one additional sulfur. Kinetic properties of purified PspE were consistent with catalysis occurring via a double-displacement mechanism via an enzyme-sulfur intermediate involving the active site cysteine. Kms for SSO32- and CN- were 2.7 mM and 32 mM, respectively, and kcat was 64s-1. The enzyme also catalyzed transfer of sulfur from thiosulfate to dithiothreitol, ultimately releasing sulfide.



Article Information


Identifiers and Pagination:

Year: 2008
Volume: 2
First Page: 18
Last Page: 28
Publisher Id: TOMICROJ-2-18
DOI: 10.2174/1874285800802010018

Article History:

Received Date: 17/1/2008
Revision Received Date: 13/2/2008
Acceptance Date: 5/3/2008
Electronic publication date: 18/3/2008
Collection year: 2008

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2008 Bentham Science Publishers Ltd

open-access license: This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.5/), which permits unrestrictive use, distribution, and reproduction in any medium, provided the original work is properly cited.


* Address correspondence to this author at the Department of Biochemistry, Virginia Polytechnic Institute and State University, Blacksburg, Virginia 24061 USA; E-mail: tilarson@vt.edu


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