Purification and characterization of S-adenosyl-L-methionine:benzoic acid carboxyl methyltransferase, the enzyme responsible for biosynthesis of the volatile ester methyl benzoate in flowers of Antirrhinum majus.
S-Adenosyl-L-methionine:benzoic acid carboxyl methyltransferase (BAMT) catalyzes the transfer of the methyl group of S-adenosyl-L-methionine (SAM) to the carboxyl group of benzoic acid to make the volatile ester methyl benzoate, one of the most abundant scent compounds of snapdragon, Antirrhinum majus. The enzyme was purified from upper and lower petal lobes of 5- to 10-day-old snapdragon flowers using DE53 anion exchange, Phenyl-Sepharose 6FF, and Mono-Q chromatography. The purified protein has a pH optimum of 7.5 and is highly specific for benzoic acid, with no activity toward several other naturally occurring substrates such as salicylic acid, cinnamic acid, and their derivatives. The molecular mass values for native and denatured protein were 100 and 49 kDa, respectively, suggesting that the active enzyme is a homodimer. The addition of monovalent cations K+ and NH4+ stimulates BAMT activity by a factor of 2, whereas the addition of Fe2+ and Cu2+ has a strong inhibitory effect. Plant-purified BAMT has Km values of 28 microM and 1.1 mM for SAM and benzoic acid, respectively (87 microM and 1.6 mM, respectively, for plant BAMT expressed in Escherichia coli). Product inhibition studies showed competitive inhibition between SAM and S-adenosyl-L-homocysteine (SAH), with a Ki of 7 microM, and noncompetitive inhibition between benzoic acid and SAH, with a Ki of 14 microM.
Murfitt
L M
LM
Department of Horticulture and Landscape Architecture, Purdue University, West Lafayette, Indiana 47907, USA.
Kolosova
N
N
Mann
C J
CJ
Dudareva
N
N
D001565:Benzoates / Q000737:chemistry* / Q000378:metabolism*; D019817:Benzoic Acid / Q000737:chemistry; D002412:Cations; D002847:Chromatography, Agarose; D002852:Chromatography, Ion Exchange; D003300:Copper / Q000737:chemistry; D019281:Dimerization; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007501:Iron / Q000737:chemistry; D007700:Kinetics; D008780:Methyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry*; D010944:Plants / Q000201:enzymology; D011188:Potassium / Q000737:chemistry; D000644:Quaternary Ammonium Compounds / Q000737:chemistry; D013696:Temperature
D001565:Benzoates; D002412:Cations; D010940:Plant Proteins; D000644:Quaternary Ammonium Compounds; C044605:methyl benzoate; D003300:Copper; D019817:Benzoic Acid; D007501:Iron; C412548:BAMT protein, Antirrhinum majus; D008780:Methyltransferases; D011188:Potassium