A eukaryotic alanine racemase gene involved in cyclic peptide biosynthesis

The cyclic tetrapeptide HC-toxin is an essential virulence determinant for the plant pathogenic fungus Cochliobolus carbonum and an inhibitor of histo ne deacetylase. The major form of RC-toxin contains the D-isomers of Ala an d Pro. The non-ribosomal peptide synthetase that synthesizes HC-toxin has o nly one epimerizing domain for conversion of L-Pro to D-Pro; the source of D-Ala has remained unknown. Here we present the cloning and characterizatio n of a new gene involved in HC-toxin biosynthesis, TOXG. TOXG is present on ly in HC-toxin-producing (Tox2(+)) isolates of C. carbonum. TOXG is able to support D-Ala-independent growth of a strain of Escherichia coli defective in D-Ala synthesis. A C. carbonum strain with both of its copies of TOXG m utated grows normally in culture, and although it no lon ger makes the thre e forms of HC-toxin that contain D-Ala, it still makes a minor form of HC-t oxin that contains Gly in place of D-Ala. The addition of D-Ala to the cult ure medium restores production of the D-Ala-containing forms of HC-toxin by the toxG mutant. The toxG mutant has only partially reduced virulence. It is concluded that TOXG encodes an alanine racemase whose function is to syn thesize D-Ala for incorporation into HC-toxin.