A role for SlyD in the Escherichia coli hydrogenase biosynthetic pathway.
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 DeltaslyD 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.
Zhang
Jie Wei
JW
Department of Chemistry, University of Toronto, Toronto, Ontario M5S 3H6, Canada.
Butland
Gareth
G
Greenblatt
Jack F
JF
Emili
Andrew
A
Zamble
Deborah B
DB
D000595:Amino Acid Sequence; D015153:Blotting, Western; D049109:Cell Proliferation; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004926:Escherichia coli / Q000235:genetics / Q000378:metabolism*; D029968:Escherichia coli Proteins / Q000737:chemistry / Q000502:physiology*; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005838:Genotype; D006864:Hydrogenase / Q000096:biosynthesis* / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D009532:Nickel / Q000737:chemistry; D010455:Peptides / Q000737:chemistry; D019696:Peptidylprolyl Isomerase / Q000502:physiology*; D010957:Plasmids / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D013997:Time Factors
D003432:Cross-Linking Reagents; D029968:Escherichia coli Proteins; D010455:Peptides; C085606:SlyD protein, E coli; D009532:Nickel; C086230:hycE protein, E coli; D006864:Hydrogenase; D019696:Peptidylprolyl Isomerase