Characterization of L-aspartate oxidase and quinolinate synthase from Bacillus subtilis.
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.
Marinoni
Ilaria
I
Department of Genetics and Microbiology, University of Pavia, Italy.
Nonnis
Simona
S
Monteferrante
Carmine
C
Heathcote
Peter
P
Härtig
Elisabeth
E
Böttger
Lars H
LH
Trautwein
Alfred X
AX
Negri
Armando
A
Albertini
Alessandra M
AM
Tedeschi
Gabriella
G
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
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