3UAO

Structure and Catalytic Mechanism of the Vitamin B3 Degradative Enzyme Maleamate Amidohydrolase from Bordetalla bronchiseptica RB50

Summary for 3UAO

• Entry DOI: 10.2210/pdb3uao/pdb
• Related: 1ILW 1IM5 1NBA 1YAC 3GBC
• Descriptor: Putative isochorismatase, ACETATE ION (3 entities in total)
• Functional Keywords: rossmann fold, hydrolase, maleamate
• Biological source: Bordetella bronchiseptica
• Total number of polymer chains: 8
• Total formula weight: 203116.64

Authors

Kincaid, V.A.,Sullivan, E.D.,Klein, R.D.,Rowlett, R.S.,Snider, M.J. (deposition date: 2011-10-21, release date: 2012-01-18, Last modification date: 2024-10-30)

Primary citation

Kincaid, V.A.,Sullivan, E.D.,Klein, R.D.,Noel, J.W.,Rowlett, R.S.,Snider, M.J.
Structure and Catalytic Mechanism of Nicotinate (Vitamin B(3)) Degradative Enzyme Maleamate Amidohydrolase from Bordetella bronchiseptica RB50.
Biochemistry, 51:545-554, 2012

PubMed Abstract: 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.

PubMed: 22214383
DOI: 10.1021/bi201347n

Experimental method

X-RAY DIFFRACTION (2.399 Å)