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.
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 2.2Å. 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 K(m) value for benzoylformate was ~23 mM, nearly 100-fold greater than that of PpBFDC while the k(cat) 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.
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Forest H
FH
Andrews
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Joshua D
JD
Horton
Department of Life Sciences, College of Natural Sciences, Sungkyunkwan University, Suwon-Si, Gyeonggi-Do 440-746, Republic of Korea.
Donghyuk
D
Shin
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Hye-Jin
HJ
Yoon
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Matthew G
MG
Logsdon
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Ahmed M
AM
Malik
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Megan P
MP
Rogers
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA.
Malea M
MM
Kneen
Department of Chemistry, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea; Department of Biophysics and Chemical Biology, College of Natural Sciences, Seoul National University, Seoul 151-742, Republic of Korea.
Se Won
SW
Suh
Department of Chemistry and Chemical Biology, Indiana University-Purdue University Indianapolis (IUPUI), Indianapolis, IN 46202, USA. Electronic address: mcleish@iupui.edu.
Michael J
MJ
McLeish
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002262:Carboxy-Lyases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D006038:Glyoxylates / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D008333:Mandelic Acids / Q000737:chemistry* / Q000378:metabolism; D020102:Mycobacterium smegmatis / Q000201:enzymology* / Q000235:genetics; D013835:Thiamine Pyrophosphate / Q000737:chemistry* / Q000378:metabolism
D001426:Bacterial Proteins; D006038:Glyoxylates; D008333:Mandelic Acids; C012482:phenylglyoxylic acid; D002262:Carboxy-Lyases; C019778:benzoylformate decarboxylase; D013835:Thiamine Pyrophosphate
Annotation; CD spectroscopy; Crystallography; Enzyme kinetics; Thiamin diphosphate