title: Structure, mechanism, and substrate profile for Sco3058: the closest bacterial homologue to human renal dipeptidase .
abstract: Human renal dipeptidase, an enzyme associated with glutathione metabolism and the hydrolysis of beta-lactams, is similar in sequence to a cluster of approximately 400 microbial proteins currently annotated as nonspecific dipeptidases within the amidohydrolase superfamily. The closest homologue to the human renal dipeptidase from a fully sequenced microbe is Sco3058 from Streptomyces coelicolor. Dipeptide substrates of Sco3058 were identified by screening a comprehensive series of l-Xaa-l-Xaa, l-Xaa-d-Xaa, and d-Xaa-l-Xaa dipeptide libraries. The substrate specificity profile shows that Sco3058 hydrolyzes a broad range of dipeptides with a marked preference for an l-amino acid at the N-terminus and a d-amino acid at the C-terminus. The best substrate identified was l-Arg-d-Asp (k(cat)/K(m) = 7.6 x 10(5) M(-1) s(-1)). The three-dimensional structure of Sco3058 was determined in the absence and presence of the inhibitors citrate and a phosphinate mimic of l-Ala-d-Asp. The enzyme folds as a (beta/alpha)(8) barrel, and two zinc ions are bound in the active site. Site-directed mutagenesis was used to probe the importance of specific residues that have direct interactions with the substrate analogues in the active site (Asp-22, His-150, Arg-223, and Asp-320). The solvent viscosity and kinetic effects of D(2)O indicate that substrate binding is relatively sticky and that proton transfers do not occurr during the rate-limiting step. A bell-shaped pH-rate profile for k(cat) and k(cat)/K(m) indicated that one group needs to be deprotonated and a second group must be protonated for optimal turnover. Computational docking of high-energy intermediate forms of l/d-Ala-l/d-Ala to the three-dimensional structure of Sco3058 identified the structural determinants for the stereochemical preferences for substrate binding and turnover.
authors.affiliation: Department of Chemistry, P.O. Box 30012, Texas A&M University, College Station, Texas 77843, USA.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Cummings
authors.forename: Tinh T
authors.initials: TT
authors.lastname: Nguyen
authors.forename: Alexander A
authors.initials: AA
authors.lastname: Fedorov
authors.forename: Peter
authors.initials: P
authors.lastname: Kolb
authors.forename: Chengfu
authors.initials: C
authors.lastname: Xu
authors.forename: Elena V
authors.initials: EV
authors.lastname: Fedorov
authors.forename: Brian K
authors.initials: BK
authors.lastname: Shoichet
authors.forename: David P
authors.initials: DP
authors.lastname: Barondeau
authors.forename: Steven C
authors.initials: SC
authors.lastname: Almo
authors.forename: Frank M
authors.initials: FM
authors.lastname: Raushel
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004150:Dipeptidases / Q000737:chemistry* / Q000378:metabolism*; D004151:Dipeptides / Q000737:chemistry / Q000378:metabolism; D006801:Humans; D048372:Streptomyces coelicolor / Q000201:enzymology / Q000378:metabolism; D013329:Structure-Activity Relationship; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D004151:Dipeptides; D004150:Dipeptidases