Study of Substrate-recognition Mechanism of Zn-metalloprotease

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The three-demensional structure and amino acid sequence of a zinc metalloprotease produced by Streptomyces caespitosus (SCNP) have been determined in order to clarify the relationship between structure and function of SCNP.The amino-acid sequence has been determined by Edman degradation. SCNP consists of a single polypeptide chain of 132 amino acid residues with one disulfide bond between residues 99 and 112. The deduced amino acid sequence indicated that it is much shorter than other ones from metalloproteases previously reported. Although a zinc-binding motif, HEXXH,found at the active sites of most metalloproteases, was found in the sequence, SCNP did not share overall significant similarity to the sequences of other zinc metalloproteases. The three-demensional structure of SCNP has been determined by X-ray crystal structure analysis and refined to R-factor of 0.18 (2.0A resolution). A five stranded beta-sheet and three alpha-helices are found in the structure. The zinc-binding moti … More f (H83-E-T-G-H87) is located on the second alpha-helix from N-terminal. About 40 amino acid sequences of zinc-containing metalloproteases have been determined so far and classified into five distinct families according to the sequence homology : thermolysin, astacin, serratia, matrixin and snake venom. Crystal structures available for six enzymes have revealed that two histidine residues located in the the sequence of HEXXH are the first two zinc ligands. Gly observed as the 8th residue from the first His in the motif, which is conserved in all but thermolysin family, has been reported to be important for structural reason, becaluse this residue allows the 11th His to be the third zinc ligand by terminating the second alpha-helix and bending a main chain sharply. SCNP also has Gly at this position. However, the residue corresponding to the 11th His is Asp. The three-dimensional structure of SCNP revealed that this Asp is the third zinc lingand. It is deduced that SCNP may represent a new subfamily of zinc-containing metalloprotease, with respect to both the new type of ligand organization for Zn and a very small molecular size distinct from other known metalloproteases in the five families. Less