Kinetic and mutational studies of three NifS homologs from Escherichia coli: mechanistic difference between L-cysteine desulfurase and L-selenocysteine lyase reactions.
We have purified three NifS homologs from Escherichia coli, CSD, CsdB, and IscS, that appear to be involved in iron-sulfur cluster formation and/or the biosynthesis of selenophosphate. All three homologs catalyze the elimination of Se and S from L-selenocysteine and L-cysteine, respectively, to form L-alanine. These pyridoxal 5'-phosphate enzymes were inactivated by abortive transamination, yielding pyruvate and a pyridoxamine 5'-phosphate form of the enzyme. The enzymes showed non-Michaelis-Menten behavior for L-selenocysteine and L-cysteine. When pyruvate was added, they showed Michaelis-Menten behavior for L-selenocysteine but not for L-cysteine. Pyruvate significantly enhanced the activity of CSD toward L-selenocysteine. Surprisingly, the enzyme activity toward L-cysteine was not increased as much by pyruvate, suggesting the presence of different rate-limiting steps or reaction mechanisms for L-cysteine desulfurization and the degradation of L-selenocysteine. We substituted Ala for each of Cys358 in CSD, Cys364 in CsdB, and Cys328 in IscS, residues that correspond to the catalytically essential Cys325 of Azotobacter vinelandii NifS. The enzyme activity toward L-cysteine was almost completely abolished by the mutations, whereas the activity toward L-selenocysteine was much less affected. This indicates that the reaction mechanism of L-cysteine desulfurization is different from that of L-selenocysteine decomposition, and that the conserved cysteine residues play a critical role only in L-cysteine desulfurization.
Mihara
H
H
Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan.
Kurihara
T
T
Yoshimura
T
T
Esaki
N
N
D001426:Bacterial Proteins / Q000378:metabolism*; D013437:Carbon-Sulfur Lyases / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000201:enzymology* / Q000235:genetics; D007700:Kinetics; D008190:Lyases / Q000235:genetics* / Q000378:metabolism; D009154:Mutation; D011732:Pyridoxal Phosphate / Q000494:pharmacology; D019289:Pyruvic Acid / Q000494:pharmacology; D017279:Selenocysteine / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity
D001426:Bacterial Proteins; C080001:nifS protein, Bacteria; D017279:Selenocysteine; D011732:Pyridoxal Phosphate; D019289:Pyruvic Acid; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; C034278:selenocysteine lyase