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Characterization of l ‐aspartate oxidase and quinolinate synthase from Bacillus subtilis
Author(s) -
Marii Ilaria,
nis Simona,
Monteferrante Carmine,
Heathcote Peter,
Härtig Elisabeth,
Böttger Lars H.,
Trautwein Alfred X.,
Negri Armando,
Albertini Alessandra M.,
Tedeschi Gabriella
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06641.x
Subject(s) - bacillus subtilis , quinolinate , nad+ kinase , biochemistry , biology , cysteine , cofactor , escherichia coli , bacteria , enzyme , atp synthase , chemistry , genetics , gene , amino acid , tryptophan , quinolinic acid
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 .