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Structural studies of maleamate amidohydrolase (NicF) from Bordetella bronchiseptica
Author(s) -
Klein Roger,
Rowlett Roger S.,
Snider Mark J.
Publication year - 2011
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.25.1_supplement.714.4
Subject(s) - amidohydrolase , tetramer , biochemistry , stereochemistry , active site , dihydroorotate dehydrogenase , chemistry , biology , enzyme
The genes coding the enzymes of aerobic catabolism of nicotinic acid in the genus Bordetella , the bacterial pathogen responsible for certain respiratory diseases in mammals, have recently been discerned. A step in this 6‐step mechanism of nicotinic acid degradation to fumaric acid is the hydrolytic deamination of maleamate to malate by maleamate amidohydrolase, NicF (EC 3.5.1.107). The BB1782 gene from Bordetella bronchiseptica was cloned, expressed in E. coli , and the resulting His6‐tagged recombinant enzyme was purified to homogeneity by affinity chromatography. The unliganded crystal structure of NicF was determined by X‐ray diffraction, using molecular replacement, to a resolution of 2.4 Å. Structural analysis predicts the biological unit to be a tetramer. The presumed catalytic triad, composed of highly conserved Lys‐Asp‐Cys, is confirmed to be within H‐bonding distance in a water‐occupied site large enough to bind maleamic acid. The N‐terminus of one subunit appears to shield the putative active site of another subunit from bulk solvent. Efforts to obtain the structure of the malate‐NicF complex are currently underway. This work was supported by NSF grant CHE‐0819686 to Colgate University and a grant to The College of Wooster from the Howard Hughes Medical Institute through the Undergraduate Science Education Program.