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...

The Escherichia coli protein SlyD is a member of the FK-506-binding protein family of peptidylprolyl isomerases. In addition to its peptidylprolyl isomerase domain, SlyD is composed of a molecular chaperone domain and a C-terminal tail rich in potential metal-binding residues. SlyD interacts with the [NiFe]-hydrogenase accessory protein HypB and contributes to nickel insertion during biosynthesis of the hydrogenase metallocenter. This study examines the HypB-SlyD complex and its significance in hydrogenase activation. Protein variants were prepared to delineate the interface between HypB and SlyD. Complex formation requires the HypB linker region located between the high affinity N-terminal Ni(II) site and the GTPase domain of the protein. In the case of SlyD, the deletion of a short loop in the chaperone domain abrogates the interaction with HypB. Mutations in either protein that disrupt complex formation in vitro also result in deficient hydrogenase production in vivo, indicating that the contact between HypB and SlyD is important for hydrogenase maturation. Surprisingly, SlyD stimulates release of nickel from the high affinity Ni(II)-binding site of HypB, an activity that is also disrupted by mutations that affect complex formation. Furthermore, a SlyD truncation lacking the C-terminal metal-binding tail still interacts with HypB but is deficient in stimulating metal release and is not functional in vivo. These results suggest that SlyD could activate metal release from HypB during metallation of the [NiFe] hydrogenase.The assembly of the [NiFe] metallocenter of Escherichia coli hydrogenase 3 requires the participation of proteins encoded by the hyp (hydrogenase pleiotropy) genes hypAB-CDEF (reviewed in Refs. 1-3). HypA and HypC are replaced by the homologous HybF and HybG proteins, respectively, for the assembly of hydrogenases 1 and 2 (1, 2). HypC, HypD, HypE, and HypF participate in the biosynthesis of the Fe(CN) 2 (CO) cluster and delivery to the hydrogenase precursor protein (4 -6). The subsequent incorporation of nickel (7,8) requires the GTPase HypB and HypA. These proteins were initially implicated in the nickel insertion step by genetic studies in which the hydrogenase deficiency resulting from chromosomal mutations was at least partially restored by growing the bacteria in excess nickel (9 -13). E. coli HypB binds one nickel ion with a K d value in the picomolar range to the cysteines in the N-terminal CXXCGC motif (referred to as the "high affinity site," see Fig. 1 for domain architecture) (14). In addition, both HypB and HypA bind a nickel ion with micromolar affinity (14 -17) as follows: HypA at a site that includes the conserved second residue His-2 (15, 16), and HypB to several conserved amino acids in the GTPase domain (referred to as the "low affinity site") (14, 18). Whether one or a combination of these metal sites serve as a source of nickel for the hydrogenase enzyme has not yet been determined.Upon searching for additional hydrogenase biosynthetic factors in E. coli, a protein called Sl...

The binding activities of IgG and IgE antibodies from egg-allergic patients to physically or chemically treated egg white proteins were examined and compared with those of rabbit anti-egg white IgG antibodies. The sera from eight patients and four rabbit antibodies were used in this study. The binding activities of human IgG antibody to partially denatured ovotransferrin (Tf), ovalbumin (OA), and lysozyme (Lys) forms were increased, whereas carboxymethylation (RCM) and heat treatment caused a dramatic decrease in the antigenicity of Tf and ovomucoid (OVM). Tf and OVM were major immunogenic antigens for the rabbit IgG response. Urea also caused Tf to exhibit greater rabbit IgG binding activity. In contrast, human and rabbit antibodies did not react with ovomucin. Partially denatured Tf and Lys also induced strong IgE binding activities. The allergenicity of Tf, OVM, and Lys was decreased by RCM, whereas OA retained its binding capacity. These results suggested that anti-OA IgE recognizes more sequential epitopes and that anti-OVM and Lys antibodies recognize both conformational and sequential epitopes. Tf and OVM were dominant allergens for the IgE antibodies of anaphylaxis patients, whereas IgE from atopic patients bound more strongly with OA and OVM.

Ovomucoid, an egg protein comprising approximately 10% egg white, was digested using the enzyme pepsin, and fragments were isolated by anion-exchange and reverse phase HPLC. Four distinct fragments were identified by analysis with SDS-PAGE, including three large fragments with molecular weights of around 24, 18, and 14 kDa. N- and C-terminal and amino acid sequencing analyses identified the fragments as V134-C186 (domain 3), V21-A133, and A1-A133 (domain 1+2). Further separation and sequencing of the fraction composed of small peptides, to determine their exact makeup and location in the protein, remained to be carried out and identified a peptide G51-Y73. All four fragments showed IgE-binding activity, as measured by ELISA, using human sera from egg-allergic individuals. Little change in the digestibility of ovomucoid by trypsin and chymotrypsin was observed following digestion with pepsin, indicating that pepsin-digested ovomucoid retains its trypsin (protease) inhibitor activities. Reduced carboxymethylated ovomucoid was prepared, and digestion with pepsin produced significantly more peptides than did the digestion of the native ovomucoid, indicating that the disulfide bonds play a significant role in the digestive resistance of ovomucoid. The reduction of ovomucoid enhanced its digestibility and lower allergenicity of the protein.