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J. Biol. Chem., Vol. 280, Issue 6, 4360-4366, February 11, 2005

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A Role for SlyD in the Escherichia coli Hydrogenase Biosynthetic Pathway^*

Jie Wei Zhang, Gareth Butland, Jack F. Greenblatt, Andrew Emili, and Deborah B. Zamble¶

From the Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada and the Banting and Best Department of Medical Research, University of Toronto, Toronto, Ontario M5G 1L6, Canada

The [NiFe] centers at the active sites of the Escherichia coli hydrogenase enzymes are assembled by a team of accessory proteins that includes the products of the hyp genes. To determine whether any other proteins are involved in this process, the sequential peptide affinity system was used. The analysis of the proteins in a complex with HypB revealed the peptidyl-prolyl cis/trans-isomerase SlyD, a metal-binding protein that has not been previously linked to the hydrogenase biosynthetic pathway. The association between HypB and SlyD was confirmed by chemical cross-linking of purified proteins. Deletion of the slyD gene resulted in a marked reduction of the hydrogenase activity in cell extracts prepared from anaerobic cultures, and an in-gel assay was used to demonstrate diminished activities of both hydrogenase 1 and 2. Western analysis revealed a decrease in the final proteolytic processing of the hydrogenase 3 HycE protein, indicating that the metal center was not assembled properly. These deficiencies were all rescued by growth in medium containing excess nickel, but zinc did not have any phenotypic effect. Experiments with radioactive nickel demonstrated that less nickel accumulated in slyD cells compared with wild type, and overexpression of SlyD from an inducible promoter doubled the level of cellular nickel. These experiments demonstrate that SlyD has a role in the nickel insertion step of the hydrogenase maturation pathway, and the possible functions of SlyD are discussed.

Received for publication, October 18, 2004 , and in revised form, November 23, 2004.

^* This work was supported in part by grants from the Natural Sciences and Engineering Research Council of Canada and the Canada Research Chairs Program (to D. B. Z.) and the Protein Engineering Network of Centers of Excellence and Genome Canada (to J. F. G. and A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^¶ To whom correspondence should be addressed. Tel./Fax: 416-978-3568; E-mail: dzamble{at}chem.utoronto.ca.

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