Functional characterization of a methionine gamma-lyase in Arabidopsis and its implication in an alternative to the reverse trans-sulfuration pathway.
Methionine gamma-lyase (MGL) catalyzes the degradation of L-methionine to alpha-ketobutyrate, methanethiol and ammonia. The Arabidopsis (Arabidopsis thaliana) genome includes a single gene (At1g64660) encoding a protein (AtMGL) with approximately 35% identity to bacterial and protozoan MGLs. When overexpressed in Escherichia coli, AtMGL allowed growth on L-methionine as sole nitrogen source and conferred a high rate of methanethiol emission. The purified recombinant protein exhibited a spectrum typical of pyridoxal 5'-phosphate enzymes, and had high activity toward l-methionine, L-ethionine, L-homocysteine and seleno-L-methionine, but not L-cysteine. Quantitation of mRNA showed that the AtMGL gene is expressed in aerial organs and roots, and that its expression in leaves was increased 2.5-fold by growth on low sulfate medium. Emission of methanethiol from Arabidopsis plants supplied with 10 mM L-methionine was undetectable (<0.5 nmol min(-1) g(-1) FW), suggesting that AtMGL is not an important source of volatile methanethiol. Knocking out the AtMGL gene significantly increased leaf methionine content (9.2-fold) and leaf and root S-methylmethionine content (4.7- and 7-fold, respectively) under conditions of sulfate starvation, indicating that AtMGL carries a significant flux in vivo. In Arabidopsis plantlets fed L-[(35)S]methionine on a low sulfate medium, label was incorporated into protein-bound cysteine as well as methionine, but incorporation into cysteine was significantly (30%) less in the knockout mutant. These data indicate that plants possess an alternative to the reverse trans-sulfuration pathway (methionine-->homocysteine-->cystathionine-->cysteine) in which methanethiol is an intermediate.
Goyer
Aymeric
A
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, USA. agoyer@pars.ars.usda.gov
Collakova
Eva
E
Shachar-Hill
Yair
Y
Hanson
Andrew D
AD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology*; D001483:Base Sequence; D013437:Carbon-Sulfur Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D017931:DNA Primers; D018076:DNA, Complementary; D004926:Escherichia coli / Q000235:genetics; D008969:Molecular Sequence Data; D009154:Mutation; D009584:Nitrogen / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction; D017386:Sequence Homology, Amino Acid; D013455:Sulfur / Q000378:metabolism*
D017931:DNA Primers; D018076:DNA, Complementary; D012333:RNA, Messenger; D013455:Sulfur; D013437:Carbon-Sulfur Lyases; C021558:L-methionine gamma-lyase; D009584:Nitrogen