Cinnamate:Coenzyme A Ligase from the Filamentous Bacterium Streptomyces coelicolor A3(2)

Abstract

4-Coumarate:coenzyme A ligase (4CL) plays a key role in phenylpropanoid metabolism, providing precursors for a large variety of important plant secondary metabolites, such as lignin, flavonoids, and phytoalexins. Although 4CLs have been believed to be specific to plants, a gene encoding a 4CL-like enzyme which shows more than 40% identity in amino acid sequence to plant 4CLs was found in the genome of the gram-positive, filamentous bacterium Streptomyces coelicolor A3(2). The recombinant enzyme, produced in Escherichia coli with a histidine tag at its N-terminal end, showed distinct 4CL activity. The optimum pH and temperature of the reaction were pH 8.0 and 30°C, respectively. The K m value for 4-coumarate and k cat were determined as 131 ؎ 4 M and 0.202 ؎ 0.007 s ؊1 , respectively. The K m value was comparable to those of plant 4CLs. The substrate specificity of this enzyme was, however, distinctly different from those of plant 4CLs. The enzyme efficiently converted cinnamate (K m , 190 ؎ 2 M; k cat , 0.475 ؎ 0.012 s ؊1 ), which is a very poor substrate for plant 4CLs. Furthermore, the enzyme showed only low activity toward caffeate and no activity toward ferulate, both of which are generally good substrates for plant 4CLs. The enzyme was therefore named ScCCL for S. coelicolor A3(2) cinnamate CoA ligase. To determine the amino acid residues providing the unique substrate specificity of ScCCL, eight ScCCL mutant enzymes having a mutation(s) at amino acid residues that probably line up along the substrate-binding pocket were generated. Mutant A294G used caffeate as a substrate more efficiently than ScCCL, and mutant A294G/A318G used ferulate, which ScCCL could not use as a substrate, suggesting that Ala 294 and Ala 318 are involved in substrate recognition. Furthermore, the catalytic activities of A294G and A294G/A318G toward cinnamate and 4-coumarate were greatly enhanced compared with those of the wild-type enzyme.The enzyme 4-coumarate:coenzyme A (CoA) ligase (4CL; EC 6.2.1.12) catalyzes the last reaction of the general phenylpropanoid pathway in plants. This pathway channels carbon flow from primary metabolism to different branch pathways of secondary phenolic metabolism via the sequential action of the enzymes phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase, and 4-coumarate:CoA ligase (4CL). PAL catalyzes the conversion of phenylalanine to cinnamate, and cinnamate 4-hydroxylase catalyzes the subsequent 4-hydroxylation of cinnamate to 4-coumarate. Finally, 4CL catalyzes the conversion of 4-coumarate (4-hydroxycinnamate) and other substituted cinnamates, such as caffeate (3,4-dihydroxycinnamate) and ferulate (3-methoxy-4-hydroxycinnamate), into the corresponding CoA thiol esters, which are used for the biosynthesis of numerous phenylpropanoid-derived compounds, such as lignins, lignans, suberins, flavonoids, isoflavonoids, and various small phenolic compounds. These compounds serve diverse functions in plants, including mechanical support and rigidity in cell walls, attractants for insect pollinat...

Combinatorial Biosynthesis of Non-bacterial and Unnatural Flavonoids, Stilbenoids and Curcuminoids by Microorganisms

“…During our mining of the "treasure trove" in Streptomyces [15], we found cinnamate/4-coumarate:CoA ligase in Streptomyces coelicolor A3(2), named ScCCL (S. coelicolor A3(2) cinnamate/4-coumarate:CoA ligase), catalyzing the conversion of both cinnamic acid (1a) and pcoumaric acid (p-hydroxycinnamate) (2a) into the corresponding CoA thiol esters 1b and 2b at almost the same efficiency [14] (Fig. 3).…”

“…This project was started in our laboratory after a type III PKS, named RppA [12,13], was for the first time discovered from the Gram-positive bacterial genus Streptomyces and its catalytic properties were elucidated. In addition, the presence of a cinnamate/4-coumarate:CoA ligase in Streptomyces [14], which is also included as a step in the phenylpropanoid pathway in plants, strongly prompted us to initiate the project. The presence of an enormous number of genes for secondary metabolite formation and modifications of exogenous substrates enabled the design of a project to produce plant-specific flavonoids in microorganisms [15].…”

Production of Plant-Specific Flavanones by Escherichia coli Containing an Artificial Gene Cluster

“…One of the barriers for the production of these compounds is the difficulty in expression of active C4H, which would not be efficiently expressed due to its instability and the lack of its specific cytochrome P-450 reductase in bacteria (8,20). We have recently discovered a 4CL in the gram-positive filamentous bacterium Streptomyces coelicolor A3(2) that can activate cinnamic acid to cinnamoyl-CoA, as well as 4-coumaric acid to 4-coumaroyl-CoA (12). The use of the bacterial 4CL enzyme would bypass the C4H step for the production of pinocembrin chalcone from phenylalanine via the phenylpropanoid pathway (Fig.…”

Metabolic Engineering of Escherichia coli for the Synthesis of the Plant Polyphenol Pinosylvin

“…However, a synthetic pathway leading to pinosylvin requires a CoA-ligase that accepts trans-cinnamic acid lacking this para-hydroxy moiety. Interestingly, Kaneko and coworkers discovered a bacterial 4CL-like enzyme from the filamentous, soil-dwelling, Gram-positive bacterium Streptomyces coelicolor (33). The enzyme showed a distinct 4CL activity, favoring trans-cinnamic acid over p-coumaric acid as the substrate.…”