STREPTOMYCES-GRISEUS AMINOPEPTIDASE - X-RAY CRYSTALLOGRAPHIC STRUCTURE AT 1.75 ANGSTROM RESOLUTION

The X-ray crystal structure of the enzyme Streptomyces griseus aminope ptidase (SGAP) has been determined in its double zinc form to 1.75 Ang strom resolution, in its apo-enzyme form (zinc removed) to 2.1 Angstro m, resolution, and as a mercury replaced derivative to 2.1 Angstrom re solution. The structure solution was achieved by single isomorphous re placement with phasing from anomalous scattering (SIRAS), followed by density modification with histogram matching. The protein consists of a central beta-sheet made up of eight parallel and antiparallel strand s, surrounded by helices on either side. The active site is located at the carbonyl ends of two middle strands of the beta-sheet region. Two sections of the chain that could not be traced were Glu196 to Arg202, which borders the active site, and the final seven C-terminal residue s starting with Gly278. The active site contains two zinc cations, eac h with similar ligands, at a distance of 3.6 Angstrom from each other. An unknown molecule appears to be bound to both zinc ions in the acti ve site at partial occupancy and has been modelled as a phosphate ion. A calcium binding site has also been identified, consistent with the observations that calcium modulates the activity of the enzyme, and in creases its heat stability. The mechanism by which the calcium cation modulates enzyme activity is not apparent, since the location of the c alcium binding site is similar to 25 Angstrom distant from the active site zinc ions. Comparison of the structure of SGAP to other known ami nopeptidases shows that the enzyme is most similar to Aeromonas proteo lytica aminopeptidase (AAP). Both enzymes share a similar topology, al though the overall sequence identity is very low (24% in aligned regio ns). The coordination of the two active site zinc cations in SGAP rese mbles that of AAP. These two microbial enzymes differ from bovine lens leucine aminopeptidase (LAP) in both overall structure and in coordin ation of the two zinc ions. (C) 1997 Academic Press Limited.