PDBsum entry 3itc

Hydrolase

PDB id: 3itc

Contents

Protein chain

392 a.a. *

Ligands

CIT

GOL

Metals

_ZN ×2

Waters ×505

* Residue conservation analysis

PDB id:

3itc

Name:

Hydrolase

Title:

Crystal structure of sco3058 with bound citrate and glycerol

Structure:

Renal dipeptidase. Chain: a. Engineered: yes

Source:

Streptomyces coelicolor. Organism_taxid: 1902. Gene: sco3058, scbac19g2.13c. Expressed in: escherichia coli. Expression_system_taxid: 562

Resolution:

1.70Å R-factor: 0.167 R-free: 0.203

Authors:

T.T.Nguyen,J.A.Cummings,C.-L.Tsai,D.P.Barondeau,F.M.Raushel

Key ref:

J.A.Cummings et al. (2010). Structure, mechanism, and substrate profile for Sco3058: the closest bacterial homologue to human renal dipeptidase . Biochemistry, 49, 611-622. PubMed id: 20000809

Date:

28-Aug-09 Release date: 02-Feb-10

Protein chain

Q93J45  (Q93J45_STRCO) -  Dipeptidase from Streptomyces coelicolor (strain ATCC BAA-471 / A3(2) / M145)

Seq: 400 a.a.

Struc: 392 a.a.

Enzyme reactions

• Enzyme class: E.C.?

Biochemistry 49:611-622 (2010)

PubMed id: 20000809

Structure, mechanism, and substrate profile for Sco3058: the closest bacterial homologue to human renal dipeptidase .

J.A.Cummings, T.T.Nguyen, A.A.Fedorov, P.Kolb, C.Xu, E.V.Fedorov, B.K.Shoichet, D.P.Barondeau, S.C.Almo, F.M.Raushel.

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.