The 1.15A crystal structure of the Staphylococcus aureus methionyl-aminopeptidase and complexes with triazole based inhibitors.

Methionyl aminopeptidases (MetAPs) represent a unique class of protease that are responsible for removing the N-terminal methionine residue from proteins and peptides. There are two major classes of MetAPs (type I and type II) described and each class can be subdivided into two subclasses. Eukaryotes contain both the type I and type II MetAPs, whereas prokaryotes possess only the type I enzyme. Due to the physiological importance of these enzymes there is considerable interest in inhibitors to be used as antiangiogenic and antimicrobial agents. Here, we describe the 1.15A crystal structure of the Staphylococcus aureus MetAP-I as an apo-enzyme and its complexes with various 1,2,4-triazole-based derivatives at high-resolution. The protein has a typical "pita-bread" fold as observed for the other MetAP structures. The inhibitors bind in the active site with the N1 and N2 atoms of the triazole moiety complexing two divalent ions. The 1,2,4-triazols represent a novel class of potent non-peptidic inhibitors for the MetAP-Is.

[1]  R. Fleischmann,et al.  Whole-genome random sequencing and assembly of Haemophilus influenzae Rd. , 1995, Science.

[2]  M. Zulauf,et al.  Light scattering of proteins as a criterion for crystallization , 1992 .

[3]  M Cascante,et al.  Relationships between inhibition constants, inhibitor concentrations for 50% inhibition and types of inhibition: new ways of analysing data. , 2001, The Biochemical journal.

[4]  Anne Techau Jørgensen,et al.  Investigation of the metal binding site in methionine aminopeptidase by density functional theory , 2002, J. Comput. Aided Mol. Des..

[5]  Ralph A. Bradshaw,et al.  N-Terminal processing: the methionine aminopeptidase and Nα-acetyl transferase families , 1998 .

[6]  X Li,et al.  Amino-terminal protein processing in Saccharomyces cerevisiae is an essential function that requires two distinct methionine aminopeptidases. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[7]  Y. Mechulam,et al.  Methionine as translation start signal: a review of the enzymes of the pathway in Escherichia coli. , 1993, Biochimie.

[8]  X. Su,et al.  Angiogenesis inhibitors specific for methionine aminopeptidase 2 as drugs for malaria and leishmaniasis. , 2002 .

[9]  D. Ingber,et al.  Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumour growth , 1990, Nature.

[10]  M. Doyle,et al.  Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2). , 2001, Biochemistry.

[11]  Paul R. Gerber,et al.  Peptide mechanics: A force field for peptides and proteins working with entire residues as smallest units , 1992 .

[12]  Thomas J. White,et al.  PCR protocols: a guide to methods and applications. , 1990 .

[13]  N. Cosper,et al.  Structural evidence that the methionyl aminopeptidase from Escherichia coli is a mononuclear metalloprotease. , 2001, Biochemistry.

[14]  O. Vallon,et al.  Control of protein life‐span by N‐terminal methionine excision , 2003, The EMBO journal.

[15]  P. Kraulis.  A program to produce both detailed and schematic plots of protein structures , 1991 .

[16]  B. Matthews,et al.  Structure and function of the methionine aminopeptidases. , 2000, Biochimica et biophysica acta.

[17]  A. Copik,et al.  Divalent metal binding properties of the methionyl aminopeptidase from Escherichia coli. , 2000, Biochemistry.

[18]  H. Grundmann,et al.  Unveiling of genetic basis of resistance of S aureus to antibiotics , 2001, The Lancet.

[19]  H. Takamatsu,et al.  Cloning and characterization of a Bacillus subtilis gene homologous to E. coli secY. , 1990, Journal of biochemistry.

[20]  R. Bradshaw,et al.  Yeast Methionine Aminopeptidase I , 1999, The Journal of Biological Chemistry.

[21]  E A Merritt,et al.  Raster3D: photorealistic molecular graphics. , 1997, Methods in enzymology.

[22]  B. Matthews,et al.  Escherichia coli methionine aminopeptidase: implications of crystallographic analyses of the native, mutant, and inhibited enzymes for the mechanism of catalysis. , 1999, Biochemistry.

[23]  R. Huber,et al.  Accurate Bond and Angle Parameters for X-ray Protein Structure Refinement , 1991 .

[24]  Insights into the mechanism of Escherichia coli methionine aminopeptidase from the structural analysis of reaction products and phosphorus-based transition-state analogues. , 1999, Biochemistry.

[25]  D. Stüber,et al.  System for High-Level Production in Escherichia coli and Rapid Purification of Recombinant Proteins: Application to Epitope Mapping, Preparation of Antibodies, and Structure—Function Analysis , 1990 .

[26]  A. Ferré-D’Amaré,et al.  Use of dynamic light scattering to assess crystallizability of macromolecules and macromolecular assemblies. , 1994, Structure.

[27]  Stephen K Burley,et al.  [10] Dynamic light scattering in evaluating crystallizability of macromolecules. , 1997, Methods in enzymology.

[28]  K. Biemann,et al.  Methionine aminopeptidase (type 2) is the common target for angiogenesis inhibitors AGM-1470 and ovalicin. , 1997, Chemistry & biology.

[29]  R A Bradshaw,et al.  Eukaryotic methionyl aminopeptidases: two classes of cobalt-dependent enzymes. , 1995, Proceedings of the National Academy of Sciences of the United States of America.

[30]  B. Matthews,et al.  The anti-angiogenic agent fumagillin covalently modifies a conserved active-site histidine in the Escherichia coli methionine aminopeptidase. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[31]  B. Matthews,et al.  Structure of the cobalt-dependent methionine aminopeptidase from Escherichia coli: a new type of proteolytic enzyme. , 1993, Biochemistry.

[32]  A. Copik,et al.  Overexpression and divalent metal binding properties of the methionyl aminopeptidase from Pyrococcus furiosus. , 2002, Biochemistry.

[33]  J. Waller,et al.  THE NH2-TERMINAL RESIDUES OF THE PROTEINS FROM CELL-FREE EXTRACTS OF E. COLI. , 1963, Journal of molecular biology.

[34]  J Navaza,et al.  Implementation of molecular replacement in AMoRe. , 2001, Acta crystallographica. Section D, Biological crystallography.

[35]  J. E. Johnston,et al.  A novel approach to crystallising proteins under oil , 1996 .

[36]  J. Smith,et al.  Molecular cloning, sequencing, deletion, and overexpression of a methionine aminopeptidase gene from Saccharomyces cerevisiae. , 1992, The Journal of biological chemistry.

[37]  K. Myambo,et al.  Processing of the initiation methionine from proteins: properties of the Escherichia coli methionine aminopeptidase and its gene structure , 1987, Journal of bacteriology.

[38]  M. Kanehisa,et al.  Whole genome sequencing of meticillin-resistant Staphylococcus aureus , 2001, The Lancet.

[39]  W. Bornmann,et al.  The anti-angiogenic agent fumagillin covalently binds and inhibits the methionine aminopeptidase, MetAP-2. , 1997, Proceedings of the National Academy of Sciences of the United States of America.

[40]  Eric C. Griffith,et al.  Molecular recognition of angiogenesis inhibitors fumagillin and ovalicin by methionine aminopeptidase 2. , 1998, Proceedings of the National Academy of Sciences of the United States of America.

[41]  C. Miller,et al.  pepM is an essential gene in Salmonella typhimurium , 1989, Journal of bacteriology.

[42]  G. Murshudov,et al.  Refinement of macromolecular structures by the maximum-likelihood method. , 1997, Acta crystallographica. Section D, Biological crystallography.

[43]  F Sherman,et al.  The specificities of yeast methionine aminopeptidase and acetylation of amino-terminal methionine in vivo. Processing of altered iso-1-cytochromes c created by oligonucleotide transformation. , 1990, The Journal of biological chemistry.

[44]  T. Yoshimoto,et al.  Two continuous spectrophotometric assays for methionine aminopeptidase. , 2000, Analytical biochemistry.

[45]  S. Chang,et al.  Methionine aminopeptidase gene of Escherichia coli is essential for cell growth , 1989, Journal of bacteriology.

[46]  N. Nomura,et al.  Prediction of the coding sequences of unidentified human genes. IV. The coding sequences of 40 new genes (KIAA0121-KIAA0160) deduced by analysis of cDNA clones from human cell line KG-1. , 1995, DNA research : an international journal for rapid publication of reports on genes and genomes.

[47]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[48]  P. Dessen,et al.  Extent of N-terminal methionine excision from Escherichia coli proteins is governed by the side-chain length of the penultimate amino acid. , 1989, Proceedings of the National Academy of Sciences of the United States of America.

[49]  J. Thornton,et al.  PROCHECK: a program to check the stereochemical quality of protein structures , 1993 .

[50]  A. D'arcy,et al.  Crystal engineering: deletion mutagenesis of the 24 kDa fragment of the DNA gyrase B subunit from Staphylococcus aureus. , 1999, Acta crystallographica. Section D, Biological crystallography.

[51]  V. D'souza,et al.  The methionyl aminopeptidase from Escherichia coli can function as an iron(II) enzyme. , 1999, Biochemistry.

[52]  J. Widom,et al.  Structure of human methionine aminopeptidase-2 complexed with fumagillin. , 1998, Science.

[53]  H. Oki,et al.  Crystal structure of methionine aminopeptidase from hyperthermophile, Pyrococcus furiosus. , 1998, Journal of molecular biology.