Abstract
5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate. EPSPSs with high catalytic efficiency and insensitivity to glyphosate are of microbial origin, including the enzyme from Agrobacterium strain CP4, in which insensitivity is conferred by an active site alanine. In the sequence context of plant EPSPSs, alanine in place of glycine at the equivalent position interferes with the binding of both glyphosate and PEP. We show here that iterative optimization of maize EPSPS containing the G101A substitution yielded variants on par with CP4 in terms of catalytic activity in the presence of glyphosate. The improvement relative to G101A alone was entirely due to reduction in Km for PEP from 333 to 18 μm, versus 9.5 μm for native maize EPSPS. A large portion of the reduction in Km was conferred by two down-sizing substitutions (L97C and V332A) within 8 Å of glyphosate, which together reduced Km for PEP to 43 μm Although the original optimization was conducted with maize EPSPS, contextually homologous substitutions conferred similar properties to the EPSPSs of other crops. We also discovered a variant having the known glyphosate-desensitizing substitution P106L plus three additional ones that reduced the Km for PEP from 47 μm, observed with P106L alone, to 10.3 μm The improvements obtained with both Ala101 and Leu106 have implications regarding glyphosate-tolerant crops and weeds.
Highlights
5-Enolpyruvylshikimate-3-phosphate synthase (EPSPS) catalyzes the transfer of a carboxyvinyl group from phosphoenolpyruvate (PEP) to shikimate-3-phosphate and in plants is the target of the herbicide glyphosate
With gene editing facilitated by Cas9/CRISPR technology [10], it is theoretically possible to create glyphosate tolerance without a foreign transgene by introducing a set of mutations into the EPSPS gene that effectively desensitizes the enzyme to glyphosate while maintaining sufficient catalytic capacity
Because the use of degenerate oligonucleotides for saturation mutagenesis theoretically can access all 19 changes at each position, we began by searching for single desensitizing mutations in native maize EPSPS that may have been missed by earlier methodology
Summary
Because the use of degenerate oligonucleotides for saturation mutagenesis theoretically can access all 19 changes at each position, we began by searching for single desensitizing mutations in native maize EPSPS that may have been missed by earlier methodology. In the context of the maize enzyme, G101A is highly insensitive to glyphosate, but has 35-fold elevated Km for PEP relative to native EPSPS (Table 2), confirming earlier results with Class I EPSPS [22, 23, 28]. Examination of a model in which the maize amino acid sequence was threaded onto PDB code 1G6S (E. coli EPSPS) provided clues as to how substitutions outside the active site could result in the dramatic reduction in Km for PEP from 333 M in G101A to 18 M in variants D2-124 and D2c-A5. The 18 mutations present in maize variant D2–124 were mapped onto the amino acid sequence of the predicted mature form of EPSPS from the species shown (see “Results” for details). The kcat of soybean D2-124 was the same as maize D2-124, but the Km for PEP was 5-fold higher, indicating that
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