Synthesis and analysis of chimeric maize glutathione transferases

Plant glutathione transferases (GSTs) are a superfamily of enzymes that have diversified to fulfil multiple functions including detoxifying xenobiotics such as herbicides, by catalysing their conjugation with glutathione. Each GST is a dimeric protein composed of subunits that each have a catalytically independent active site derived from an N-terminal glutathione binding domain (G-site) and a C-terminal hydrophobic binding domain (H-site). We have been interested in generating novel xenobiotic-detoxifying enzymes by combining domains from different GSTs. Using herbicide-detoxifying maize (Zea mays L.) phi (F) and tau (U) classes of GSTs, we have generated and expressed in Escherichia coli a series of intra- and inter-class chimeric enzymes by combining different G- and H-sites. Soluble, active chimeric GSTs could be generated between members of each class with the resulting enzymes showing novel substrate binding characteristics. In contrast, inter-class chimeras were all expressed as insoluble inactive protein aggregates. Analysis of representative GSTU and GSTF crystal structures suggested that this was due to disrupted dimer formation rather than any major perturbation in monomer folding. Studies with the intra-class GSTU chimeras were extended to generate mutant enzymes derived from a range of splice sites throughout the gene. These mainly possessed hybrid activities individually determined by each H- and G-module but unexpectedly the H-site also influenced glutathione binding.