(1) Department of Inorganic Chemistry and the Laboratory of Structural Chemistry
and Biology, The Hebrew University of Jerusalem, Jerusalem 91904, Israel.
(2) Dipartimento di Scienze Biochimiche 'A. Rossi Fanelli' and Centro di Biologia
Molecolare del Consiglio Nazionale delle Ricerche, Universi La Sapienza, 00185
Roma, Italy.
(3) Sackler Institute of Molecular Medicine, Sackler Faculty of Medicine, Tel
Aviv University, Tel Aviv 69978, Israel.
The three dimensional crystal structure of the double-zinc aminopeptidase from Streptomyces griseus (SGAP) has been determined to 1.75Å resolution. The solution of the structure was achieved by single isomorphous replacement with phasing from anomalous scattering (SIRAS), followed by density modification with histogram matching. The SGAP structure belongs to the general class of alpha/beta proteins. It consists of a central beta-sheet which is made up of 8 parallel and anti-parallel strands, surrounded on either side by a total of 9 alpha-helices. The active site contains two zinc cations at a distance of 3.6Å from each other and bridged by two shared ligands. Each of the zinc ions is pentacoordinated with similar ligands and geometry. An unknown molecule appears to be bound to both zinc ions in the active site at partial occupancy. This has been modeled as a phosphate ion, based on the shape of the density and hydrogen bonding patterns. 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 this dication modulates enzyme activity is not apparent, since the location of the calcium binding site is ~25Å distant from the active site zinc ions. Data from one crystal of the native enzyme indicated partial oxidation of one of the methionine residues to a sulfoxide, an oxidation that was attributed to the age of the crystal. The structure of the apo-SGAP, where both zinc ions and the calcium ion has been removed, has also been solved to 2.1Å resolution. This structure showed rather little conformational changes in the overall structure and around the zinc and calcium sites, and indicated that neither type of ions play significant structural role. Apo-SGAP was used to prepare a derivative of the native protein where the two active site zinc ions were replaced with a single mercury ion. The structure of this metallo- derivative were solved to 2.1Å resolution and suggested that the two catalytic metal sites of the native enzyme are not equivalent. 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. The two enzymes differ from the high molecular weight bovine lens leucine aminopeptidase (LAP) in their overall structure and in the coordination pattern of the two zinc ions.