Interactions of Streptomyces griseus aminopeptidase with a methionine product analogue: a structural study at 1.53 angstrom resolution

SGAP is an aminopeptidase present in the extracellular fluid of Streptomyce s 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 c alcium ions. The small size, high activity and heat stability make SGAP a v ery attractive enzyme for various biotechnological applications. Only one o ther related aminopeptidase (Aeromonas proteolytica AP; AAP) has been struc turally analyzed to date and its structure was shown to be considerably sim ilar to SGAP, despite the low sequence homology between the two enzymes. Th e 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 ngstrom crystallographic structure of native SGAP has been previously repor ted, but did not allow critical mechanistic interpretations owing to inconc lusive structural regions around the active site. A more accurate structure of SGAP at 1.58 Angstrom resolution is reported in this paper, along with the 1.53 Angstrom resolution structure of the SGAP complex with inhibitory methionine, which is also a product of the SGAP catalytic process. These tw o high-resolution structures enable a better understanding of the SGAP bind ing 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 invo lved in enzyme-substrate interactions (Asp160, Met161, Gly201, Arg202 and P he219). These studies also suggest that Glu131 is directly involved in the catalytic mechanism of SGAP, probably as the hydrolytic nucleophile. The st ructural results are compared with a recent structure of AAP with an hydrox amate inhibitor in order to draw general functional conclusions which are r elevant for this family of low molecular-weight aminopeptidases.