Characterization of IMT1, myo-inositol O-methyltransferase, from Mesembryanthemum crystallinum.
A full-length transcript, Imt1, encoding myo-inositol O-methyltransferase (EC 2.1.1.X) from the halophyte Mesembryanthemum crystallinum was expressed in Escherichia coli. The enzyme, IMT1, uses S-adenosyl-L-methionine to methylate myo-inositol to form D-ononitol. IMT1 with a monomeric mass of 41,000 was isolated by ammonium sulfate fractionation, gel filtration and ion exchange chromatography to apparent purity on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of the purified recombinant enzyme was identical to that encoded by the cDNA sequence. The apparent Km for S-adenosylmethionine was 0.18 mM with a Vmax of 1550 pkat/mg protein. The Km for myo-inositol was 1.32 mM. The reaction became substrate-inhibited by concentrations of S-adenosylmethionine greater than 0.5 mM. Inositol methyltransferase was competitively inhibited 50% with 0.01 mM S-adenosyl-homocysteine, while 1 mM homocysteine, homoserine, or adenosine did not inhibit. The enzyme exhibited a pH optimum of 7.8 and a temperature optimum of 37 degrees C. Activity of the isolated inositol methyltransferase was stable when stored at 4 degrees C.
Rammesmayer
G
G
Department of Biochemistry, University of Arizona, Tucson 85721, USA.
Pichorner
H
H
Adams
P
P
Jensen
R G
RG
Bohnert
H J
HJ
D000595:Amino Acid Sequence; D018744:DNA, Plant / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D007294:Inositol / Q000031:analogs & derivatives / Q000737:chemistry; D007700:Kinetics; D008780:Methyltransferases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008969:Molecular Sequence Data; D008970:Molecular Weight; D010944:Plants / Q000201:enzymology* / Q000235:genetics; D011994:Recombinant Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D012435:S-Adenosylhomocysteine / Q000378:metabolism; D012436:S-Adenosylmethionine / Q000378:metabolism; D013237:Stereoisomerism; D013379:Substrate Specificity
D018744:DNA, Plant; D011994:Recombinant Proteins; D007294:Inositol; D012436:S-Adenosylmethionine; D012435:S-Adenosylhomocysteine; D008780:Methyltransferases; C095694:inositol O-methyltransferase