Anthranilate N-methyltransferase, a branch-point enzyme of acridone biosynthesis.
Acridone alkaloids formed by acridone synthase in Ruta graveolens L. are composed of N-methylanthraniloyl CoA and malonyl CoAs. A 1095 bp cDNA from elicited Ruta cells was expressed in Escherichia coli, and shown to encode S-adenosyl-l-methionine-dependent anthranilate N-methyltransferase. SDS-PAGE of the purified enzyme revealed a mass of 40 +/- 2 kDa, corresponding to 40 059 Da for the translated polypeptide, whereas the catalytic activity was assigned to a homodimer. Alignments revealed closest relationships to catechol or caffeate O-methyltransferases at 56% and 55% identity (73% similarity), respectively, with little similarity ( approximately 20%) to N-methyltransferases for purines, putrescine, glycine, or nicotinic acid substrates. Notably, a single Asn residue replacing Glu that is conserved in caffeate O-methyltransferases determines the catalytic efficiency. The recombinant enzyme showed narrow specificity for anthranilate, and did not methylate catechol, salicylate, caffeate, or 3- and 4-aminobenzoate. Moreover, anthraniloyl CoA was not accepted. As Ruta graveolens acridone synthase also does not accept anthraniloyl CoA as a starter substrate, the anthranilate N-methylation prior to CoA activation is a key step in acridone alkaloid formation, channelling anthranilate from primary into secondary branch pathways, and holds promise for biotechnological applications. RT-PCR amplifications and Western blotting revealed expression of the N-methyltransferase in all organs of Ruta plants, particularly in the flower and root, mainly associated with vascular tissues. This expression correlated with the pattern reported previously for expression of acridone synthase and acridone alkaloid accumulation.
Rohde
Britta
B
Institut für Pharmazeutische Biologie, Philipps-Universität Marburg, Deutschhausstrasse 17A, 35037 Marburg, Germany.
Hans
Joachim
J
Martens
Stefan
S
Baumert
Alfred
A
Hunziker
Peter
P
Matern
Ulrich
U
D010129:4-Aminobenzoic Acid / Q000378:metabolism; D000166:Acridines / Q000378:metabolism*; D054831:Acridones; D000595:Amino Acid Sequence; D015153:Blotting, Western; D002396:Catechols / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D016133:Polymerase Chain Reaction; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D027821:Rutaceae / Q000235:genetics / Q000378:metabolism*; D012459:Salicylates / Q000378:metabolism; D013379:Substrate Specificity; D062367:ortho-Aminobenzoates / Q000378:metabolism*
D000166:Acridines; D054831:Acridones; D002396:Catechols; D010940:Plant Proteins; D011994:Recombinant Proteins; D012459:Salicylates; D062367:ortho-Aminobenzoates; C031385:anthranilic acid; C041300:acridone; D008780:Methyltransferases; C034221:catechol; D010129:4-Aminobenzoic Acid