No abstract

Streptomyces griseus aminopeptidase (SGAP) is a double-zinc exopeptidase with a high preference toward large hydrophobic amino-terminus residues. It is a monomer of a relatively low molecular weight (30 kDa), it is heat stable, it displays a high and efficient catalytic turnover, and its activity is modulated by calcium ions. The small size, high activity, and heat stability make SGAP a very attractive enzyme for various biotechnological applications, among which is the processing of recombinant DNA proteins and fusion protein products. Several free amino acids, such as phenylalanine, leucine, and methionine, were found to act as weak inhibitors of SGAP and hence were chosen for structural studies. These inhibitors can potentially be regarded as product analogs because one of the products obtained in a normal enzymatic reaction is the cleaved amino terminal amino acid of the substrate. The current study includes the X-ray crystallographic analysis of the SGAP complexes with methionine (1.53 A resolution), leucine (1.70 A resolution), and phenylalanine (1.80 A resolution). These three high-resolution structures have been used to fully characterize the SGAP active site and to identify some of the functional groups of the enzyme that are involved in enzyme-substrate and enzyme-product interactions. A unique binding site for the terminal amine group of the substrate (including the side chains of Glu131 and Asp160, as well as the carbonyl group of Arg202) is indicated to play an important role in the binding and orientation of both the substrate and the product of the catalytic reaction. These studies also suggest that Glu131 and Tyr246 are directly involved in the catalytic mechanism of the enzyme. Both of these residues seem to be important for substrate binding and orientation, as well as the stabilization of the tetrahedral transition state of the enzyme-substrate complex. Glu131 is specifically suggested to function as a general base during catalysis by promoting the nucleophilic attack of the zinc-bound water/hydroxide on the substrate carbonyl carbon. The structures of the three SGAP complexes are compared with recent structures of three related aminopeptidases: Aeromonas proteolytica aminopeptidase (AAP), leucine aminopeptidase (LAP), and methionine aminopeptidase (MAP) and their complexes with corresponding inhibitors and analogs. These structural results have been used for the simulation of several species along the reaction coordinate and for the suggestion of a general scheme for the proteolytic reaction catalyzed by SGAP.

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyces griseus cultures. It is a double-zinc enzyme with a strong preference for large hydrophobic amino-terminus residues. It is a monomeric (30 kDa) heat-stable enzyme, with a high and efficient catalytic activity modulated by calcium ions. The small size, high activity and heat stability make SGAP a very attractive enzyme for various biotechnological applications. Only one other related aminopeptidase (Aeromonas proteolytica AP; AAP) has been structurally analyzed to date and its structure was shown to be considerably similar to SGAP, despite the low sequence homology between the two enzymes. The motivation for the detailed structural analysis of SGAP originated from a strong mechanistic interest in the family of double-zinc aminopeptidases, combined with the high potential applicability of these enzymes. The 1.75 A crystallographic structure of native SGAP has been previously reported, but did not allow critical mechanistic interpretations owing to inconclusive structural regions around the active site. A more accurate structure of SGAP at 1.58 A resolution is reported in this paper, along with the 1.53 A resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These two high-resolution structures enable a better understanding of the SGAP binding mode of both substrates and products. These studies allowed the tracing of the previously disordered region of the enzyme (Glu196-Arg202) and the identification of some of the functional groups of the enzyme that are involved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and Phe219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The structural results are compared with a recent structure of AAP with an hydroxamate inhibitor in order to draw general functional conclusions which are relevant for this family of low molecular-weight aminopeptidases.

We determined the prevalence and risk factors of H. pylori infection among 197 healthy 3- to 5-year-old Israeli Arab children, in a population under socioeconomic and environmental transition. Data on the socioeconomic and environmental characteristics were obtained by personal interviews. The presence of H. pylori infection was identified using an ELISA kit for detection of H. pylori antigens in stool specimens. The prevalence rate of H. pylori infection was 49.7% (95% CI 42.8-56.67). It varied significantly among the different villages. In the univariate analysis stratified by village, the risk of infection increased according to household crowding, number of siblings younger than 5 years and siblings' H. pylori positivity. In the multivariate analysis the village of residence and siblings' H. pylori positivity were the only variables that remained strongly associated with H. pylori infection. In a population such as that described in this study the socioeconomic and living conditions are major risk factors of H. pylori infection and the intra-familial transmission of H. pylori in early childhood has an important role.

The aminopeptidase from Streptomyces griseus is a calcium-activated metalloenzyme, which contains 2 mol tightly bound zinc/mol protein. This aminopeptidase rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids, such as leucine, methionine and phenylalanine. We have determined the complete primary structure of the protein, which contains 284 amino acid residues, yielding a molecular mass of 29723 Da. A search in the Swiss-Prot database for sequence similarities revealed a low degree of identity (26 -34 %) to Succharornyces cerevisiae aminopeptidase Y, Aeromonas proteolytica aminopeptidase, and a hypothetical 49.5-kDa protein from Bacillus subtilis, which is supposed to belong to the aminopeptidase Y family. In all these proteins, the residues that are known to be involved in zinc coordination are conserved.Keywords: metalloproteins ; Streptomyces griseus ; zinc-containing aminopeptidases ; primary structure.Aminopeptidase from Streptomyces griseus (S. griseus AP) is a calcium-activated zinc metalloprotein, characterized by high enzyme activity, high thermal stability, and low molecular mass [I -31. The enzyme contains 2 mol tightly bound zinc/mol protein and it can bind calcium concomitantly with an increase in activity [3, 41. S. griseus AP rapidly hydrolyzes peptide bonds formed by N-terminal hydrophobic amino acids such as leucine, methionine and phenylalanine. Due to its unique physico-chemical properties and high enzymatic activity, the use of S. griseus AP has been proposed as a tool to attack peptide and protein substrates in a variety of biochemical and biotechnological applications [3 -51. Due to the presence of two zinc atoms/molecule, the enzyme is thought to resemble one or more of the other aminopeptidases with two transition metal atoms/molecule or subunit, like Aeromoms proreolytica aminopeptidase (A. proteolyticn AP) [6), methionyl aminopeptidase 171 or leucyl aminopeptidase [8], whereas it is expected to differ from aminopeptidases with one zinc atom at their catalytic site, like aminopeptidase N [9]. The occurrence of a presumed additional modulatory site for calcium binding is another interesting structural and functional feature of S. griseus AP, which renders the enzyme a most attractive target for structural and mechanistic investigations of metallopeptidases.