Tryptophan Synthase from Agrobacterium tumefaciens 8628: Isolation and Properties

N. I. Rekoslavskaya,^1,2 E. V. Kuznetsova,¹ E. F. Vysotskaya,¹ and R. K. Salyaev¹

¹Siberian Institute of Plant Physiology and Biochemistry, Siberian Branch of the Russian Academy of Sciences, Irkutsk, P. O. Box 1243, 664033 Russia; fax: (3952) 310754; E-mail: root@sifibr.irkutsk.su

²To whom correspondence should be addressed.

Submitted January 20, 1996; revision submitted January 20, 1997.

Tryptophan synthase was isolated from a highly virulent strain of Agrobacterium tumefaciens 8628 (octopine type). Separation of tryptophan synthase from thermolabile protease was accomplished using fractionation with polyethylene glycol-6000 followed by ion-exchange chromatography with a pH gradient. Molecular weights of alpha- and beta-subunits are 33 and 51 kD, respectively. The tryptophan synthase is stable at 60°C because of heat-tolerate beta-subunits. After heating the activity of tryptophan synthase increased up to 20 times while temperature-labile proteases lost their activities. Reaction with antibodies showed the presence of four protein bands, one of which was coeluted with nucleic acids during ion-exchange chromatography. It is suggested that the basic tryptophan synthase is encoded by trp genes in a plasmid and its role is to provide the precursor with the prokaryotic pathway of indole-3-acetic acid biosynthesis, which determines the virulence of A. tumefaciens. There is perhaps a cooperation between iaaM, iaaH, and trp genes in the plasmid during plant cell transformation.

KEY WORDS: Agrobacterium tumefaciens, transformation, tryptophan synthase.

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