Dissecting the indigo pathway

Indigo is a vibrant blue pigment synthesised by plants and bacteria. This study examined the mechanisms by which plants generate indole, an intermediate common to both the indigo and tryptophan biosynthetic pathways. Cleavage of Indole-3-glycerol phosphate (I3GP) by the tryptophan synthase (TS) αβ enzyme complex forms indole for subsequent tryptophan biosynthesis. Specifically, indole synthesis is catalysed by the TSα sub-unit, encoded by the trpA gene, leading to this sub-unit being described as an ‘indole synthase’. Typically, TSα sub-units only catalyse indole synthesis when bound to a specific TSβ sub-unit counterpart. However, there does exist in plants enzymes capable of catalysing indole synthesis independently. In Zea mays, there is an indole synthase (IS) enzyme ten times more efficient than the TSαβ enzyme complex at catalysing indole biosynthesis from I3GP. When the maize indole synthase gene was expressed in a TSα deficient E. coli strain it complemented the trpA mutation, indicating that the enzyme encoded by this gene produced indole as an intermediate in tryptophan biosynthesis in the absence of a plant specific TSβ sub-unit. The removal of tryptophanase activity by targeted mutagenesis in a host E. coli strain eliminated the alternative route for tryptophan biosynthesis from indole. Complementation by the Z. mays IS of the trpA mutation suggests that the IS forms a functional enzyme complex with an E. coli TSβ sub-unit. A Polygonum tinctorium cDNA library was screened via hybridisation with the Z. mays IS gene and by functional analysis. The unsuccessful hybridisation suggested the absence of a gene homologous to the maize IS gene within P. tinctorium, whilst failure of the cDNA library to rescue the E. coli trpA mutant strain indicated the non-existance of a functional clone