Mechanistic Characterization of Escherichia coli l -Aspartate Oxidase from Kinetic Isotope Effects

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Abstract

l-Aspartate oxidase, encoded by the nadB gene, is the first enzyme in the de novo synthesis of NAD+ in bacteria. This FAD-dependent enzyme catalyzes the oxidation of l-aspartate to generate iminoaspartate and reduced flavin. Distinct from most amino acid oxidases, it can use either molecular oxygen or fumarate to reoxidize the reduced enzyme. Sequence alignments and the three-dimensional crystal structure have revealed that the overall fold and catalytic residues of NadB closely resemble those of the succinate dehydrogenase/fumarate reductase family rather than those of the prototypical d-amino acid oxidases. This suggests that the enzyme can catalyze amino acid oxidation via typical amino acid oxidase chemistry, involving the removal of protons from the α-amino group and the transfer of the hydride from C2, or potentially deprotonation at C3 followed by transfer of the hydride from C2, similar to chemistry occurring during succinate oxidation. We have investigated this potential mechanistic ambiguity using a combination of primary, solvent, and multiple deuterium kinetic isotope effects in steady state experiments. Our results indicate that the chemistry is similar to that of typical amino acid oxidases in which the transfer of the hydride from C2 of l-aspartate to FAD is rate-limiting and occurs in a concerted manner with respect to deprotonation of the α-amine. Together with previous kinetic and structural data, we propose that NadB has structurally evolved from succinate dehydrogenase/fumarate reductase-type enzymes to gain the new functionality of oxidizing amino acids while retaining the ability to reduce fumarate.

Original languageEnglish (US)
Pages (from-to)4044-4052
Number of pages9
JournalBiochemistry
Volume56
Issue number31
DOIs
StatePublished - Aug 8 2017

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Aspartic Acid
Isotopes
Escherichia coli
Oxidoreductases
Succinate Dehydrogenase
Amino Acids
Kinetics
Hydrides
Enzymes
Fumarates
Deprotonation
Flavin-Adenine Dinucleotide
Oxidation
Molecular oxygen
Sequence Alignment
Deuterium
Succinic Acid
NAD
Amines
Protons

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanistic Characterization of Escherichia coli l -Aspartate Oxidase from Kinetic Isotope Effects. / Chow, Carmen; Hegde, Subray; Blanchard, John S.

In: Biochemistry, Vol. 56, No. 31, 08.08.2017, p. 4044-4052.

Research output: Contribution to journalArticle

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