Characterization of a NifS-like chloroplast protein from Arabidopsis. Implications for its role in sulfur and selenium metabolism.
NifS-like proteins catalyze the formation of elemental sulfur (S) and alanine from cysteine (Cys) or of elemental selenium (Se) and alanine from seleno-Cys. Cys desulfurase activity is required to produce the S of iron (Fe)-S clusters, whereas seleno-Cys lyase activity is needed for the incorporation of Se in selenoproteins. In plants, the chloroplast is the location of (seleno) Cys formation and a location of Fe-S cluster formation. The goal of these studies was to identify and characterize chloroplast NifS-like proteins. Using seleno-Cys as a substrate, it was found that 25% to 30% of the NifS activity in green tissue in Arabidopsis is present in chloroplasts. A cDNA encoding a putative chloroplast NifS-like protein, AtCpNifS, was cloned, and its chloroplast localization was confirmed using immunoblot analysis and in vitro import. AtCpNIFS is expressed in all major tissue types. The protein was expressed in Escherichia coli and purified. The enzyme contains a pyridoxal 5' phosphate cofactor and is a dimer. It is a type II NifS-like protein, more similar to bacterial seleno-Cys lyases than to Cys desulfurases. The enzyme is active on both seleno-Cys and Cys but has a much higher activity toward the Se substrate. The possible role of AtCpNifS in plastidic Fe-S cluster formation or in Se metabolism is discussed.
Pilon-Smits
Elizabeth A H
EA
Biology Department, Colorado State University, Fort Collins, Colorado 80523, USA. epsmits@lamar.colostate.edu
Garifullina
Gulnara F
GF
Abdel-Ghany
Salah
S
Kato
Shin-Ichiro
S
Mihara
Hisaaki
H
Hale
Kerry L
KL
Burkhead
Jason L
JL
Esaki
Nobuyoshi
N
Kurihara
Tatsuo
T
Pilon
Marinus
M
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000235:genetics / Q000378:metabolism*; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D013437:Carbon-Sulfur Lyases; D002736:Chloroplasts / Q000378:metabolism*; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000235:genetics; D008190:Lyases / Q000378:metabolism; D008969:Molecular Sequence Data; D009586:Nitrogen Fixation / Q000235:genetics; D010802:Phylogeny; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D012643:Selenium / Q000378:metabolism*; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*
D029681:Arabidopsis Proteins; C472234:At1g08490 protein, Arabidopsis; D018076:DNA, Complementary; D012333:RNA, Messenger; D013455:Sulfur; D008190:Lyases; D013437:Carbon-Sulfur Lyases; C080546:cysteine desulfurase; C034278:selenocysteine lyase; D012643:Selenium