A Role for SlyD in the Escherichia coli Hydrogenase Biosynthetic Pathway

Abstract

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.The production of metalloenzymes frequently requires dedicated auxiliary proteins to assemble the functional metallocenters (1, 2). In the case of an enzyme with a single ion bound to unmodified protein ligands, maturation usually involves just one partner protein (1, 3). These factors, referred to as metallochaperones (3), deliver the correct metal ion to the target protein via protein-protein interactions (1). For the biosynthesis of more complex metallocenters, multiple accessory proteins are often required (2). These factors control a cascade of events that can include gathering and insertion of all of the inorganic and organic components, partial construction of the metal center, posttranslational modifications, electron transfer, protein folding, and/or hydrolysis of nucleotide triphosphates to drive the whole process forward (2). These molecular factories generate enzymes that are essential for a variety of fundamental cellular processes, but many of the protein components have not yet been identified or fully characterized.The hydrogenase enzymes, which catalyze the reversible formation of dihydrogen (H 2 ) from two protons and two electrons, contain several different types of active sites (4, 5). In Escherichia coli the hydrogenases are all members of the [NiFe] class of enzymes that have nickel, iron, and three non-protein diatomic ligands in a deeply buried active site (6, 7). The outline of the general sequence of events during hydrogenase metallocenter assembly in E. coli has been largely derived from studies of the hydro...

Hydrogenase activity in the foodborne pathogen Campylobacter jejuni depends upon a novel ABC-type nickel transporter (NikZYXWV) and is SlyD-independent

“…However, like H. pylori, C. jejuni does encode a SlyD homologue (Cj0115). In E. coli, mutation of slyD results in lower intracellular nickel, failure to process the large subunit of hydrogenase 3 (HycE) and thus decreased hydrogenase activity (Zhang et al, 2005). These mutant phenotypes were complemented by excess nickel, which suggested that SlyD had a role in nickel insertion into the apohydrogenase rather than preceding steps.…”

“…These mutant phenotypes were complemented by excess nickel, which suggested that SlyD had a role in nickel insertion into the apohydrogenase rather than preceding steps. This is supported by the interaction of SlyD with the hydrogenase accessory protein HypB (Zhang et al, 2005), possibly as a trigger of metal release (Leach et al, 2007). We therefore hypothesized that mutation of slyD in C. jejuni may result in decreased intracellular nickel and a concomitant reduction in hydrogenase activity.…”

Properties of the [NiFe]‐hydrogenase maturation protein HypD

“…The core machinery, as demonstrated for the synthesis of the intracellularly located hydrogenase 3 from E. coli, consists of the products of the six hyp genes plus an endopeptidase ( [1][2][3][4] and for review see [5,6]). The two hyp gene products HypA and HypB together with the auxiliary protein SlyD [7][8][9] are required for nickel sequestration and incorporation, whereas the four other Hyp proteins (HypC to HypF) are involved in synthesis of the CN ligand and the cyanation of the active site iron [10][11][12][13]. The source and biosynthesis of the CO ligand are still unknown [14].…”

Metal Selectivity of the Escherichia coli Nickel Metallochaperone, SlyD

“…SlyD contributes to both nickel accumulation and energy metabolism in this organism because it participates in the Ni(II) insertion step during [NiFe]-hydrogenase metallocenter assembly (4,5). NMR solution structures of E. coli SlyD revealed that the N-terminal region consists of two well-defined domains (6,7).…”