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Anthranilate N-methyltransferase, a branch-point enzyme of acridone biosynthesis


Rohde, B; Hans, J; Martens, S; Baumert, A; Hunziker, P; Matern, U (2008). Anthranilate N-methyltransferase, a branch-point enzyme of acridone biosynthesis. The Plant Journal, 53(3):541-553.

Abstract

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.

Abstract

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.

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Additional indexing

Item Type:Journal Article, refereed, original work
Communities & Collections:04 Faculty of Medicine > Functional Genomics Center Zurich
08 University Research Priority Programs > Systems Biology / Functional Genomics
Dewey Decimal Classification:570 Life sciences; biology
610 Medicine & health
Language:English
Date:February 2008
Deposited On:06 Jan 2010 13:18
Last Modified:05 Apr 2016 13:36
Publisher:Wiley-Blackwell
ISSN:0960-7412
Free access at:Publisher DOI. An embargo period may apply.
Publisher DOI:https://doi.org/10.1111/j.1365-313X.2007.03360.x
PubMed ID:17988223

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