Interaction of the hydrogenase accessory protein HypC with HycE, the large subunit of Escherichia coli hydrogenase 3 during enzyme maturation.
Maturation of the large subunit of E. coli hydrogenase 3, HycE, requires the action of seven accessory proteins. The HycI protease catalyses a C-terminal proteolytic cleavage of the large subunit, which was shown to result in a dramatic change in migration behavior of HycE in nondenaturing PAGE. HypA, HypB, HypC, HypD, HypE, and HypF are required for metallocenter assembly. A polyacrylamide gel system under nondenaturing conditions was used for the investigation of any protein-protein interactions between HycE and the Hyp proteins. It revealed the existence of a complex between the precursor of HycE (pre-HycE) with one of the accessory proteins, namely HypC. HypC migrates in at least three different forms in nondenaturing PAGE, the appearance of one of which (form 1) is strictly dependent on the presence of unprocessed HycE in the extract. Overexpression of either hypC or hycE from a plasmid leads to an increased formation of this HypC-form 1. In two-dimensional polyacrylamide gel electrophoresis with nondenaturing PAGE as the first and SDS-PAGE as the second dimension, this HypC form comigrates with part of the pre-HycE protein. This comigration was also observed in anion exchange chromatography. To analyze the pre-HycE-HypC complex in more detail, HypC was overproduced and purified. The purified protein was able to bind to pre-HycE in vitro. These results and also the finding that the processed form of HycE is not associated with HypC suggest that HypC binds to pre-HycE to keep it in a conformation accessible for metal incorporation.
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Lehrstuhl fur Mikrobiologie der Universitat Munchen, Maria-Ward-Strasse 1a, 80638 Munchen, Germany.
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D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D015180:Electrophoresis, Gel, Two-Dimensional; D004926:Escherichia coli / Q000201:enzymology; D029968:Escherichia coli Proteins; D006864:Hydrogenase / Q000378:metabolism*; D011487:Protein Conformation; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; C108874:HypC protein, Bacteria; C491587:HypC protein, E coli; C086230:hycE protein, E coli; D006864:Hydrogenase