Please note these web pages are part of an assignment for a graduate course in Advanced Biochemistry and Molecular Biology BCMB8010 at the University of Georgia. Questions should be directed to Gina Pries (gpries@uga.edu).

Introduction

The monomeric enzyme 5-enolpyruvylshikimate-3-phosphate (EPSP) synthase (EPSPS) is one of two enzymes in the class of enolpyruvyltransferases. This class shares a unique structure containing two globular domains composed of beta sheets and alpha helices which form something like an inverse alpha/beta barrel. The two domains are connected by two strands which act like a hinge to bring the upper and lower domains together, sandwiching the substrates in the active site (1). Ligand binding converts the enzyme from an open state to a tightly-packed closed state, following the pattern of an induced-fit mechanism (1,2). EPSP synthase is involved in the shikimate pathway, using phosphoenolpyruvate (PEP) to convert shikimate-3-phosphate (S3P) to 5-enolpyruvyl-3-shikimate phosphate (Fig. 1), a precursor to the majority of aromatic compounds produced in the cell, including the aromatic amino acids. It has been reported that the compounds produced in this pathway constitute as much as 35% or more of the dry mass of plants (3). The fact that this enzyme does not occur in mammals, fish, reptiles, birds and insects makes it a good target for antibiotics (4) and herbicides (5). The synthetic compound glyphosate (N-phosphonomethylglycine, the active ingredient in the herbicides Roundup™ (6) and Touchdown™) is a competitive inhibitor to this enzyme (Fig. 2) (7), effectively shutting down aromatic amino acid biosynthesis and also synthesis of other aromatic compounds derived from these amino acids. The agricultural benefit comes with genetically engineered plants which are resistant to the herbicide (8); Roundup-ready crops may be treated to eliminate weeds and other problem plants while leaving the engineered plants unharmed. Research indicates that glyphosate acts as transition-state analogue (9), effectively shutting down the Shikimate pathway. In this way, glyphosate deregulates feedback inhibition of the first enzyme in the pathway by a near-end product of the pathway (10), and as a result, there is an unregulated flow of carbon into the pathway causing high levels of S3P to accumulate (11). Glyphosate displays high specificity for EPSP synthase, not even binding to UDP-N-acetylglucosamine enolpyruvyltransferase (MurA) (12), the only other enzyme in the enolpyruvyltransferase class. The purpose of this report is to summarize the function and importance of EPSP sythase and its interaction with substrates and the synthetic inhibitor glyphosate as well as to detail the structural aspects of these relationships.

Abreviations used: EPSP, 5-enolpyruvylshikimate-3-phosphate [EC 2.5.1.19]; PEP, phosphoenolpyruvate; S3P, shikimate-3-phosphate; MurA, UDP-N-acetylglucosamine enolpyruvyltransferase [EC 2.5.1.7]; EPSPS, 5-enolpyruvylshikimate-3-phosphate synthase.