Streptomyces griseus aminopeptidase: X-ray crystallographic structure at 1.75 A resolution.

Abstract

The X-ray crystal structure of the enzyme Streptomyces griseus aminopeptidase (SGAP) has been determined in its double zinc form to 1.75 A resolution, in its apo-enzyme from (zinc removed) to 2.1 A resolution, and as a mercury replaced derivative to 2.1 A resolution. The structure solution was achieved by single isomorphous replacement 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 strands, 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 residues starting with Gly278. The active site contains two zinc cations, each with similar ligands, at a distance of 3.6 A from each other. An unknown molecule appears to be bound to both zinc ions in the active 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 increases its heat stability. The mechanism by which the calcium cation modulates enzyme activity is not apparent, since the location of the calcium binding site is approximately 25 A distant from the active site zinc ions. Comparison of the structure of SGAP to other known aminopeptidases shows that the enzyme is most similar to Aeromonas proteolytica aminopeptidase (AAP). Both enzymes share a similar topology, although the overall sequence identity is very low (24% in aligned regions). The coordination of the two active site zinc cations in SGAP resembles that of AAP. These two microbial enzymes differ from bovine lens leucine aminopeptidase (LAP) in both overall structure and in coordination of the two zinc ions.