Biochemical analysis of a thermostable tryptophan synthase from a hyperthermophilic archaeon.

Pyridoxal 5'-phosphate-dependent tryptophan synthase catalyzes the last two reactions of tryptophan biosynthesis, and is comprised of two distinct subunits, alpha and beta. TktrpA and TktrpB, which encode the alpha subunit and beta subunit of tryptophan synthase from a hyperthermophilic archaeon, Thermococcus kodakaraensis KOD1, were independently expressed in Escherichia coli and their protein products were purified. Tryptophan synthase complex (Tk-TS complex), obtained by heat treatment of a mixture of the cell-free extracts containing each subunit, was also purified. Gel-filtration chromatography revealed that Tk-TrpA was a monomer (alpha), Tk-TrpB was a dimer (beta2), and Tk-TS complex was a tetramer (alpha2 beta2). The Tk-TS complex catalyzed the overall alphabeta reaction with a specific activity of 110 micromol Trp per micromol active site per min under its optimal conditions (80 degrees C, pH 8.5). Individual activity of the alpha and beta reactions of the Tk-TS complex were 8.5 micromol indole per micromol active site per min (70 degrees C, pH 7.0) and 119 micromol Trp per micromol active site per min (90 degrees C, pH 7.0), respectively. The low activity of the alpha reaction of the Tk-TS complex indicated that turnover of the beta reaction, namely the consumption of indole, was necessary for efficient progression of the alpha reaction. The alpha and beta reaction activities of independently purified Tk-TrpA and Tk-TrpB were 10-fold lower than the respective activities detected from the Tk-TS complex, indicating that during heat treatment, each subunit was necessary for the other to obtain a proper conformation for high enzyme activity. Tk-TrpA showed only trace activities at all temperatures examined (40-85 degrees C). Tk-TrpB also displayed low levels of activity at temperatures below 70 degrees C. However, Tk-TrpB activity increased at temperatures above 70 degrees C, and eventually at 100 degrees C, reached an equivalent level of activity with the beta reaction activity of Tk-TS complex. Taking into account the results of circular dichroism analyses of the three enzymes, a model is proposed which explains the relationship between structure and activity of the alpha and beta subunits with changes in temperature. This is the first report of an archaeal tryptophan synthase, and the first biochemical analysis of a thermostable tryptophan synthase at high temperature.