Arabidopsis thaliana tryptophan synthase alpha: gene cloning, expression, and subunit interaction.

The tryptophan synthase alpha subunit catalyzes the conversion of indole-3-glycerolphosphate to indole, the penultimate reaction in the biosynthesis of the essential amino acid tryptophan. A cDNA encoding Arabidopsis thaliana tryptophan synthase alpha(TSA1) was isolated by complementation of an Escherichia coli delta trpA mutation and by polymerase chain reaction amplification from a cDNA library using degenerate primers. A TSA1 genomic clone was also isolated and 5 kb of the DNA sequence determined. A single sequence in the Arabidopsis genome with homology to the TSA1 cDNA was detected by high-stringency genomic Southern blot hybridization. In contrast under hybridization conditions of reduced stringency, one or two additional homologous sequences were observed. A 1.4 kb transcript was detected in wild-type RNA with the TSA1 cDNA as a probe. Several lines of evidence, including immunoaffinity chromatography, suggest that the active A. thaliana tryptophan synthase enzyme consists of a heterosubunit complex, presumably analogous to the prokaryotic alpha 2 beta 2 complex. Immunoblot analysis indicated that the plant alpha and beta subunits are present throughout development.