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Journal of Biochemistry Advance Access published online on October 15, 2007
Journal of Biochemistry, 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

Kazuki Fukuma1, Evan D. Neuls2, Jennifer M. Ryberg2, Dae-Yeon Suh2,* and Ushio Sankawa1,3

1 Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama 930-0194, Japan; 2 Department of Chemistry and Biochemistry, University of Regina, Regina, SK S4S 0A2, Canada; and 3 International Traditional Medicine Research Center, Toyama International Health Complex, Toyama 939-8224, Japan.

*To whom correspondence should be addressed: Prof. Dae-Yeon Suh, Tel: +1-306-585-4239, Fax: +1-306-337-2409, E-mail: suhdaey{at}uregina.ca

Received August 18, 2007; Accepted September 24, 2007


  Abstract

Chalcone synthase (CHS), a key enzyme in flavonoid biosynthesis, catalyzes 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 three-dimensional 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 catalyzes 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, stilbene synthase, type III polyketide synthase, outer sphere mutation, site-directed mutagenesis


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