Structure and catalytic mechanism of nicotinate (vitamin B3) degradative enzyme maleamate amidohydrolase from Bordetella bronchiseptica RB50.
The penultimate reaction in the oxidative degradation of nicotinate (vitamin B(3)) to fumarate in several species of aerobic bacteria is the hydrolytic deamination of maleamate to maleate, catalyzed by maleamate amidohydrolase (NicF). Although it has been considered a model system for bacterial degradation of N-heterocyclic compounds, only recently have gene clusters that encode the enzymes of this catabolic pathway been identified to allow detailed investigations concerning the structural basis of their mechanisms. Here, the Bb1774 gene from Bordetella bronchiseptica RB50, putatively annotated as nicF, has been cloned, and the recombinant enzyme, overexpressed and purified from Escherichia coli, is shown to catalyze efficiently the hydrolysis of maleamate to maleate and ammonium ion. Steady-state kinetic analysis of the reaction by isothermal titration calorimetry (ITC) established k(cat) and K(M) values (pH 7.5 and 25 °C) of 11.7 ± 0.2 s(-1) and 128 ± 6 μM, respectively. The observed K(D) of the NicF·maleate (E·P) complex, also measured by ITC, is approximated to be 3.8 ± 0.4 mM. The crystal structure of NicF, determined at 2.4 Å using molecular replacement, shows that the enzyme belongs to the cysteine hydrolase superfamily. The structure provides insight concerning the roles of potential catalytically important residues, most notably a conserved catalytic triad (Asp29, Lys117, and Cys150) observed in the proximity of a conserved non-proline cis-peptide bond within a small cavity that is likely the active site. On the basis of this structural information, the hydrolysis of maleamate is proposed to proceed by a nucleophilic addition-elimination sequence involving the thiolate side chain of Cys150.
Department of Chemistry, The College of Wooster, Wooster, Ohio 44691, United States.
Virginia A
VA
Kincaid
Eric D
ED
Sullivan
Roger D
RD
Klein
Jeff W
JW
Noel
Roger S
RS
Rowlett
Mark J
MJ
Snider
D000595:Amino Acid Sequence; D000641:Ammonia / Q000737:chemistry* / Q000378:metabolism; D016950:Bordetella bronchiseptica / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics; D015964:Gene Expression Regulation, Bacterial; D006868:Hydrolysis; D008298:Maleates / Q000737:chemistry* / Q000378:metabolism; D008969:Molecular Sequence Data; D009525:Niacin / Q000737:chemistry; D009535:Nicotinamidase / Q000737:chemistry* / Q000235:genetics / Q000502:physiology; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification; D013329:Structure-Activity Relationship
D008298:Maleates; D011994:Recombinant Proteins; D009525:Niacin; D000641:Ammonia; D009535:Nicotinamidase; C008311:maleamic acid