Structure, mechanism, and substrate profile for Sco3058

The closest bacterial homologue to human renal dipeptidase

Jennifer A. Cummings, Tinh T. Nguyen, Alexander A. Fedorov, Peter Kolb, Chengfu Xu, Elena V. Fedorov, Brian K. Shoichet, David P. Barondeau, Steven C. Almo, Frank M. Raushel

Research output: Contribution to journalArticle

13 Citations (Scopus)

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 x 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). The solvent viscosity and kinetic effects of 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.

Original languageEnglish (US)
Pages (from-to)611-622
Number of pages12
JournalBiochemistry
Volume49
Issue number3
DOIs
StatePublished - Jan 26 2010

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dipeptidase 1
Dipeptides
Dipeptidases
Catalytic Domain
Substrates
Amidohydrolases
Streptomyces coelicolor
L Forms
Amino Acids
Lactams
Enzymes
Substrate Specificity
Site-Directed Mutagenesis
Viscosity
Citric Acid
Glutathione
Protons
Zinc
Hydrolysis
Ions

ASJC Scopus subject areas

  • Biochemistry

Cite this

Cummings, J. A., Nguyen, T. T., Fedorov, A. A., Kolb, P., Xu, C., Fedorov, E. V., ... Raushel, F. M. (2010). Structure, mechanism, and substrate profile for Sco3058: The closest bacterial homologue to human renal dipeptidase. Biochemistry, 49(3), 611-622. https://doi.org/10.1021/bi901935y

Structure, mechanism, and substrate profile for Sco3058 : The closest bacterial homologue to human renal dipeptidase. / Cummings, Jennifer A.; Nguyen, Tinh T.; Fedorov, Alexander A.; Kolb, Peter; Xu, Chengfu; Fedorov, Elena V.; Shoichet, Brian K.; Barondeau, David P.; Almo, Steven C.; Raushel, Frank M.

In: Biochemistry, Vol. 49, No. 3, 26.01.2010, p. 611-622.

Research output: Contribution to journalArticle

Cummings, JA, Nguyen, TT, Fedorov, AA, Kolb, P, Xu, C, Fedorov, EV, Shoichet, BK, Barondeau, DP, Almo, SC & Raushel, FM 2010, 'Structure, mechanism, and substrate profile for Sco3058: The closest bacterial homologue to human renal dipeptidase', Biochemistry, vol. 49, no. 3, pp. 611-622. https://doi.org/10.1021/bi901935y
Cummings, Jennifer A. ; Nguyen, Tinh T. ; Fedorov, Alexander A. ; Kolb, Peter ; Xu, Chengfu ; Fedorov, Elena V. ; Shoichet, Brian K. ; Barondeau, David P. ; Almo, Steven C. ; Raushel, Frank M. / Structure, mechanism, and substrate profile for Sco3058 : The closest bacterial homologue to human renal dipeptidase. In: Biochemistry. 2010 ; Vol. 49, No. 3. pp. 611-622.
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AU - Kolb, Peter

AU - Xu, Chengfu

AU - Fedorov, Elena V.

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