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 β-lactams, is similar in sequence to a cluster of ~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 (kcat/Km = 7.6 × 105 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 (β/α)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). Solvent viscosity and kinetic effects by D2O 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 kcat and kcat/Km 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.

The Creatininase Homolog MftE from Mycobacterium smegmatis Catalyzes a Peptide Cleavage Reaction in the Biosynthesis of a Novel Ribosomally Synthesized Post-translationally Modified Peptide (RiPP)

“…Members of the amidohydrolase superfamily catalyze hydrolysis of a diverse array of substrates (26). Within the vast superfamily, there are examples of enzymes, such as renal dipeptidase, that catalyze the hydrolysis of peptide amide bonds (27,28). However, to our knowledge, MftE is unique in catalyzing the hydrolysis of a peptide bond in a 30-amino acid modified protein.…”

The solution of nitrogen inversion in amidases

“…In analogy to the zinc proteases an Asp or Glu general base participates in the reaction mechanism. Sco3058 is a bacterial dipeptidase that displays substrate specificity for d ‐amino acids on the C‐terminal side of the scissile bond . Sco3058 displays an enzyme assisted hydrogen bond in the TS accepted by the backbone of G323 (Fig.…”

Nontherapeutic equivalence of a generic product of imipenem-cilastatin is caused more by chemical instability of the active pharmaceutical ingredient (imipenem) than by its substandard amount of cilastatin

“…Although P . aeruginosa ATCC 27853 is a wild-type strain, all members of this species produce a membrane dipeptidase called CdhR (a transcriptional regulator of carnitine catabolic genes) that, belonging to the M19 family and being homologue of the mammalian DHP-I, could have hydrolytic activity against carbapenems [40–42].…”