The kinetic characterization and X-ray structure of a putative benzoylformate decarboxylase from M. smegmatis highlights the difficulties in the functional annotation of ThDP-dependent enzymes

Forest H. Andrews, Joshua D. Horton, Donghyuk Shin, Hye Jin Yoon, Matthew G. Logsdon, Ahmed M. Malik, Megan P. Rogers, Malea M. Kneen, Se Won Suh, Michael J. McLeish

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Abstract

Abstract Benzoylformate decarboxylase (BFDC) is a thiamin diphosphate (ThDP)-dependent enzyme that catalyzes the nonoxidative decarboxylation of benzoylformate. It is the penultimate enzyme in both the mandelate pathway and the d-phenylglycine degradation pathway. The ThDP-dependent Enzyme Engineering Database (TEED) now lists more than 800 sequences annotated as BFDCs, including one from Mycobacterium smegmatis (MsBFDC). However, there is no evidence that either pathway for benzoylformate formation exists in the M. smegmatis genome. Further, sequence alignments of MsBFDC with the well characterized enzyme isolated from Pseudomonas putida (PpBFDC) indicate that there will be active site substitutions in MsBFDC likely to reduce activity with benzoylformate. Taken together these data would suggest that the annotation is unlikely to be correct. To test this hypothesis the putative MsBFDC was cloned, expressed, purified, and the X-ray structure was solved to a resolution of 2.2 Å. While showing no evidence for ThDP in the active site, the structure was very similar to that of PpBFDC. A number of 2-oxo acids were tested as substrates. For MsBFDC the Km value for benzoylformate was ~ 23 mM, nearly 100-fold greater than that of PpBFDC while the kcat value was reduced 60-fold. These values would suggest that benzoylformate is not the physiological substrate for this enzyme, and that annotation as a 2-oxo acid decarboxylase may be more appropriate.

Original languageEnglish
Article number39590
Pages (from-to)1001-1009
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1854
Issue number8
DOIs
Publication statusPublished - 2015 Jul 1

Bibliographical note

Funding Information:
This work was supported in part by an East Asia Pacific Summer Fellowship co-sponsored by the National Science Foundation (NSF) and the National Research Council of Korea (to F.H.A.), NSF Grant CHE 1307877 (M.J.M.) and the IUPUI Center for Research and Learning Undergraduate Research Opportunity Program (J.D.H., M.G.L., A.M.M.). We thank the beamline staff at Pohang Light Source, Korea (BL-5C) for their assistance during X-ray diffraction experiments.

Publisher Copyright:
© 2015 Elsevier B.V.

All Science Journal Classification (ASJC) codes

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

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