Structural and Mutational Studies of Anthocyanin Malonyltransferases Establish the Features of BAHD Enzyme Catalysis

Hideaki Unno,Hirokazu Suzuki,Masami Kusunoki,Atsushi Saito,Tokuzo Nishino,Toru Nakayama,Seiji Takahashi,Yoshikazu Tanaka,Fumiko Ichimaida

doi:10.1074/jbc.m700638200

Hideaki Unno, Hirokazu Suzuki + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m700638200

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Abstract

The BAHD family is a class of acyl-CoA-dependent acyltransferases that are involved in plant secondary metabolism and show a diverse range of specificities for acyl acceptors. Anthocyanin acyltransferases make up an important class of the BAHD family and catalyze the acylation of anthocyanins that are responsible for most of the red-to-blue colors of flowers. Here, we describe crystallographic and mutational studies of three similar anthocyanin malonyltransferases from red chrysanthemum petals: anthocyanidin 3-O-glucoside-6''-O-malonyltransferase (Dm3MaT1), anthocyanidin 3-O-glucoside-3'', 6''-O-dimalonyltransferase (Dm3MaT2), and a homolog (Dm3MaT3). Mutational analyses revealed that seven amino acid residues in the N- and C-terminal regions are important for the differential acyl-acceptor specificity between Dm3MaT1 and Dm3MaT2. Crystallographic studies of Dm3MaT3 provided the first structure of a BAHD member, complexed with acyl-CoA, showing the detailed interactions between the enzyme and acyl-CoA molecules. The structure, combined with the results of mutational analyses, allowed us to identify the acyl-acceptor binding site of anthocyanin malonyltransferases, which is structurally different from the corresponding portion of vinorine synthase, another BAHD member, thus permitting the diversity of the acyl-acceptor specificity of BAHD family to be understood.

Highlights

Transferases plays versatile roles in the biosyntheses of these metabolites, making a very important contribution to the establishment of the structural and functional diversities of the metabolites [1]
It has been proposed that Anthocyanin acyltransferases (AATs) catalysis proceeds through the formation of a ternary complex consisting of acyl-CoA, an anthocyanin, and the enzyme, where a general-base amino acid residue deprotonates a hydroxy group of the anthocyanin substrate, thereby promoting its nucleophilic attack on the carbonyl of the thioester of acyl-CoA [6]
Region Swapping and Site-specific Mutagenesis of Dm3MaT1 and Dm3MaT2—To explore the amino acid residues responsible for the difference in the acyl-acceptor specificity of malonyl transfer between Dm3MaT1 and Dm3MaT2, we first carried out region swapping between these enzymes

Summary

Enzyme Assay

The standard reaction mixture (50 ␮l) consisted of 20 mM potassium phosphate, pH 7.0, 60 ␮M malonyl-CoA, 120 ␮M cyanidin 3-O-. Dm3MaT1 shows exclusive 3MaT1 520 nm; flow rate, 0.7 ml/min. It is noteworthy that these two malonyltransferases (MaTs) are 89% identical to each other in their primary structures [8], serving as very good targets for studies of structural factors that govern the acyl acceptor specificity of AATs. developed with 20% B for 3 min followed by a linear gradient from 20 to 31% B in 15 min followed by that from 31 to 56% B in 1 min. The first crystal structure of a free enzyme form of a BAHD family member, vinorine synthase of the medicinal plant Rauvolfia serpentina, was reported in 2005 [9], the crystal structure of a BAHD member complexed with acyl-CoA has not been reported.

Enzyme Kinetics

RESULTS AND DISCUSSION

Space group