Characterization and metabolic function of a peroxisomal sarcosine and pipecolate oxidase from Arabidopsis.
Sarcosine oxidase (SOX) is known as a peroxisomal enzyme in mammals and as a sarcosine-inducible enzyme in soil bacteria. Its presence in plants was unsuspected until the Arabidopsis genome was found to encode a protein (AtSOX) with approximately 33% sequence identity to mammalian and bacterial SOXs. When overexpressed in Escherichia coli, AtSOX enhanced growth on sarcosine as sole nitrogen source, showing that it has SOX activity in vivo, and the recombinant protein catalyzed the oxidation of sarcosine to glycine, formaldehyde, and H(2) O(2) in vitro. AtSOX also attacked other N-methyl amino acids and, like mammalian SOXs, catalyzed the oxidation of l-pipecolate to Delta(1)-piperideine-6-carboxylate. Like bacterial monomeric SOXs, AtSOX was active as a monomer, contained FAD covalently bound to a cysteine residue near the C terminus, and was not stimulated by tetrahydrofolate. Although AtSOX lacks a typical peroxisome-targeting signal, in vitro assays established that it is imported into peroxisomes. Quantitation of mRNA showed that AtSOX is expressed at a low level throughout the plant and is not sarcosine-inducible. Consistent with a low level of AtSOX expression, Arabidopsis plantlets slowly metabolized supplied [(14)C]sarcosine to glycine and serine. Gas chromatography-mass spectrometry analysis revealed low levels of pipecolate but almost no sarcosine in wild type Arabidopsis and showed that pipecolate but not sarcosine accumulated 6-fold when AtSOX expression was suppressed by RNA interference. Moreover, the pipecolate catabolite alpha-aminoadipate decreased 30-fold in RNA interference plants. These data indicate that pipecolate is the endogenous substrate for SOX in plants and that plants can utilize exogenous sarcosine opportunistically, sarcosine being a common soil metabolite.
Goyer
Aymeric
A
Horticultural Sciences Department, University of Florida, Gainesville, Florida 32611, USA.
Johnson
Tanya L
TL
Olsen
Laura J
LJ
Collakova
Eva
E
Shachar-Hill
Yair
Y
Rhodes
David
D
Hanson
Andrew D
AD
D000595:Amino Acid Sequence; D017360:Arabidopsis / Q000201:enzymology* / Q000378:metabolism*; D028464:Cucurbita / Q000378:metabolism; D003545:Cysteine / Q000737:chemistry; D018076:DNA, Complementary / Q000378:metabolism; D004926:Escherichia coli / Q000378:metabolism; D005557:Formaldehyde / Q000737:chemistry; D008401:Gas Chromatography-Mass Spectrometry; D005998:Glycine / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000737:chemistry; D013058:Mass Spectrometry; D008830:Microbodies / Q000378:metabolism; D008956:Models, Chemical; D008969:Molecular Sequence Data; D009584:Nitrogen / Q000737:chemistry / Q000378:metabolism; D000587:Oxidoreductases Acting on CH-NH Group Donors / Q000737:chemistry*; D010100:Oxygen / Q000378:metabolism; D020675:Peroxisomes / Q000737:chemistry / Q000201:enzymology* / Q000378:metabolism; D017434:Protein Structure, Tertiary; D034622:RNA Interference; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000737:chemistry; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012521:Sarcosine / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013763:Tetrahydrofolates / Q000494:pharmacology
D018076:DNA, Complementary; D012333:RNA, Messenger; D011994:Recombinant Proteins; D013763:Tetrahydrofolates; D005557:Formaldehyde; C030371:5,6,7,8-tetrahydrofolic acid; D012694:Serine; D006861:Hydrogen Peroxide; D000587:Oxidoreductases Acting on CH-NH Group Donors; C032771:L-pipecolate dehydrogenase; D003545:Cysteine; D009584:Nitrogen; D010100:Oxygen; D005998:Glycine; D012521:Sarcosine