Analysis of the cleavage site specificity of the endopeptidase involved in the maturation of the large subunit of hydrogenase 3 from Escherichia coli.
The maturation of [NiFe]-hydrogenases is a catalysed process in which the activities of at least seven proteins are involved. The last step consists of the endoproteolytic cleavage of the precursor of the large subunit after the [NiFe]-metal centre has been assembled. The amino acid sequence requirements for the endopeptidase HycI involved in the C-terminal processing of HycE, the large subunit of the hydrogenase 3 from Escherichia coli, were investigated. Mutational alteration of the amino acid residues neighbouring the cleavage site showed that proteolysis still occurred when chemically similar amino acids were exchanged. Processing was blocked, however, in a variant in which the methionine at the C-terminal side was replaced by a glutamate residue. Truncation of the precursor from the C-terminal end rendered variants amenable to maturation even when two-thirds of the extension were removed but abolished proteolysis upon further deletion of a cluster of six basic amino acids. A construct in which the C-terminal extension from the large subunit of the hydrogenase 2 was fused to the mature part of the large subunit of hydrogenase 3 was neither processed by HycI nor by HybD, the endopeptidase specific for the large subunit of hydrogenase 2. The maturation endopeptidase, therefore, exhibits a relaxed sequence constraint in recognition of its cleavage site and does not require the entire C-terminal extension. The results point to an interaction of the C-terminus with some domain of the large subunit, rendering a conformation amenable to recognition by the endopeptidase.
Theodoratou
E
E
Institut für Genetik und Mikrobiologie, Universität München, Munich, Germany.
Paschos
A
A
Mintz-Weber
Böck
A
A
D000595:Amino Acid Sequence; D010450:Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics / Q000254:growth & development; D029968:Escherichia coli Proteins; D006864:Hydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009154:Mutation
D029968:Escherichia coli Proteins; C086230:hycE protein, E coli; D006864:Hydrogenase; D010450:Endopeptidases