Journal of Biochemistry Advance Access originally published online on October 15, 2007
Journal of Biochemistry 2007 142(6):731-739; doi:10.1093/jb/mvm188
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© 2007 The Japanese Biochemical Society.
Mutational Analysis of Conserved Outer Sphere Arginine Residues of Chalcone Synthase
1Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan; 2Department of Chemistry and Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada; and 3International Traditional Medicine Research Center, Toyama International Health Complex, Toyama 939-8224, Japan
*To whom correspondence should be addressed. Tel: +1-306-585-4239, Fax: +1-306-337-2409, E-mail: suhdaey{at}uregina.ca
Received August 18, 2007; Accepted September 24, 2007
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Abstract |
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Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyses sequential decarboxylative condensations of p-coumaroyl-CoA with three malonyl-CoA molecules and cyclizes the resulting tetraketide intermediate to produce chalcone. Phenylglyoxal, an Arg selective reagent, was found to inactivate the enzyme, although no Arg is found at the active site. Conserved, non-active site Arg residues of CHS were individually mutated and the results were discussed in the context of the 3D structure of CHS. Arg199 and Arg350 were shown to provide important interactions to maintain the structural integrity and foldability of the enzyme. Arg68, Arg172 and Arg328 interact with highly conserved Gln33/Phe215, Glu380 and Asp311/Glu314, respectively, thus helping position the catalytic Cys-His-Asn triad and the 372GFGPG loop in correct topology at the active site. In particular, a mutation of Arg172 resulted in selective impairment in the cyclization activities of CHS and stilbene synthase, a related enzyme that catalyses a different cyclization of the same tetraketide intermediate. These Arg residues and their interactions are well conserved in other enzymes of the CHS superfamily, suggesting that they may serve similar functions in other enzymes. Mutations of Arg68 and Arg328 had been found in mutant plants that showed impaired CHS activity.
Key Words: chalcone synthase, outer sphere mutation, stilbene synthase, type III polyketide synthase, site-directed mutagenesis
Abbreviations: 2-PS, 2-pyrone synthase; BNY, bisnoryangonin; CHS, chalcone synthase; CTAL, p-coumaroyltriacetic acid lactone; CTAS, coumaroyltriacetic acid synthase; KAS, β-ketoacyl-acyl carrier protein synthase; PCS, pentaketide chromone synthase; PGO, phenylglyoxal; PKS, polyketide synthase; STS, stilbene synthase; THNS, 1,3,6,8-tetrahydroxynaphthalene synthase