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

H. M. Greenblatt, O. Almog, B. Maras, A. Spungin-Bialik, D. Barra, S. Blumberg, G. Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

113 Scopus citations

Abstract

The X-ray crystal structure of the enzyme Streptomyces griseus aminopeptidase (SGAP) has been determined in its double zinc form to 1.75 Å resolution, in its apo-enzyme form (zinc removed) to 2.1 Å resolution, and as a mercury replaced derivative to 2.1 Å 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 β-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 β-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 Å 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 ~ 25 Å 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.

Original languageAmerican English
Pages (from-to)620-636
Number of pages17
JournalJournal of Molecular Biology
Volume265
Issue number5
DOIs
StatePublished - 7 Jan 1997

Bibliographical note

Funding Information:
We thank E. Dodson for her advice and assistance with the CCP4 programs during the course of the structure elucidation. H.M.G. thanks A. J. Bauer for helpful advice. We thank the National Synchrotron Light Source (NSLS) and the Biology Department of the Brookhaven National Laboratory for beam time at the X12B beamline. We acknowledge V. Stojanoff of BNL for her critical assistance with X-ray data collection at the NSLS. We also thank D. Schomburg for helpful discussions and preliminary molecular weight determination of SGAP by electron spray mass spectroscopy. This work was supported in part by a grant to G.S. and S.B. from the Ministry of Science and the Arts of Israel, and the Gesellschaft für Biotechnologische Forschung, MBH, Braunschweig, Germany, and in part by a grant to B.M. and D.B. from the Italian Ministero dell’Università della Ricerca Scientifica e Tecnologica. G.S. thanks The Friends of the Hebrew University in England and Ms Myrtle Franklin for financial support. H.M.G. thanks the Canadian Friends of the Hebrew University, the Morris M. Pulver Foundation, and Corob Holdings for partial financial support.

Keywords

  • Aminopeptidase
  • Anomalous scattering
  • Calcium activation
  • Crystal structure
  • Double-zinc metalloproteinase

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