title: Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis.
abstract: NAD is an important cofactor and essential molecule in all living organisms. In many eubacteria, including several pathogens, the first two steps in the de novo synthesis of NAD are catalyzed by l-aspartate oxidase (NadB) and quinolinate synthase (NadA). Despite the important role played by these two enzymes in NAD metabolism, many of their biochemical and structural properties are still largely unknown. In the present study, we cloned, overexpressed and characterized NadA and NadB from Bacillus subtilis, one of the best studied bacteria and a model organism for low-GC Gram-positive bacteria. Our data demonstrated that NadA from B. subtilis possesses a [4Fe-4S]2+ cluster, and we also identified the cysteine residues involved in the cluster binding. The [4Fe-4S]2+ cluster is coordinated by three cysteine residues (Cys110, Cys230, and Cys320) that are conserved in all the NadA sequences reported so far, suggesting a new noncanonical binding motif that, on the basis of sequence alignment studies, may be common to other quinolinate synthases from different organisms. Moreover, for the first time, it was shown that the interaction between NadA and NadB is not species-specific between B. subtilis and Escherichia coli.
authors.affiliation: Department of Genetics and Microbiology, University of Pavia, Italy.
authors.forename: Ilaria
authors.initials: I
authors.lastname: Marinoni
authors.forename: Simona
authors.initials: S
authors.lastname: Nonnis
authors.forename: Carmine
authors.initials: C
authors.lastname: Monteferrante
authors.forename: Peter
authors.initials: P
authors.lastname: Heathcote
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Härtig
authors.forename: Lars H
authors.initials: LH
authors.lastname: Böttger
authors.forename: Alfred X
authors.initials: AX
authors.lastname: Trautwein
authors.forename: Armando
authors.initials: A
authors.lastname: Negri
authors.forename: Alessandra M
authors.initials: AM
authors.lastname: Albertini
authors.forename: Gabriella
authors.initials: G
authors.lastname: Tedeschi
mesh_terms: D000594:Amino Acid Oxidoreductases / Q000737:chemistry* / Q000235:genetics; D001412:Bacillus subtilis / Q000201:enzymology*; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics; D001665:Binding Sites; D003001:Cloning, Molecular; D017124:Conserved Sequence; D003545:Cysteine; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes / Q000737:chemistry* / Q000235:genetics; D013045:Species Specificity
chemical_list: D001426:Bacterial Proteins; D029968:Escherichia coli Proteins; D007506:Iron-Sulfur Proteins; D009097:Multienzyme Complexes; C022512:quinolinic acid synthetase; D000594:Amino Acid Oxidoreductases; C031955:L-aspartate oxidase, E coli; D003545:Cysteine