Following evolution's lead to a single residue switch for diterpene synthase product outcome

Abstract

There have been few insights into the biochemical origins of natural product biosynthesis from primary metabolism. Of particular interest are terpene synthases, which often mediate the committed step in particular biosynthetic pathways so that alteration of their product outcome is a key step in the derivation of novel natural products. These enzymes also catalyze complex reactions of significant mechanistic interest. Following an evolutionary lead from two recently diverged, functionally distinct diterpene synthase orthologs from different subspecies of rice, we have identified a single residue that can switch product outcome. Specifically, the mutation of a conserved isoleucine to threonine that acts to convert not only the originally targeted isokaurene synthase into a specific pimaradiene synthase but also has a much broader effect, which includes conversion of the ent-kaurene synthases found in all higher plants for gibberellin phytohormone biosynthesis to the production of pimaradiene. This surprisingly facile switch for diterpene synthase catalytic specificity indicates the ease with which primary (gibberellin) metabolism can be subverted to secondary biosynthesis and may underlie the widespread occurrence of pimaradiene-derived natural products. In addition, because this isoleucine is required for the mechanistically more complex cyclization to tetracyclic kaurene, whereas substitution with threonine ''short-circuits'' this mechanism to produce the ''simpler'' tricyclic pimaradiene, our results have some implications regarding the means by which terpene synthases specify product outcome.enzyme specificity ͉ natural product biosynthesis ͉ terpene synthase ͉ biochemical evolution T he evolution of secondary metabolism presumably originates via changes in the catalytic specificity of enzymes recruited from primary metabolism. Although a recent report demonstrates that small numbers of changes in a sesquiterpene synthase can dramatically shift product outcome (i.e., plasticity), the target enzyme was already involved in secondary metabolism, and the parent wild-type enzyme was quite promiscuous (i.e., produced many different products) (1). Thus, how readily the typically specific enzymes involved in primary metabolism can be subverted into secondary metabolism remains a matter of conjecture.Terpene synthases carry out complex electrophilic cyclization/ rearrangement reactions, creating diverse hydrocarbon skeletal structures from simpler isoprenoid precursors, which often represents the committed step in particular biosynthetic pathways. Hence, altering the function of these enzymes is expected to represent a key step in the evolution of secondary metabolism. In addition, terpene synthases have attracted a great deal of interest because of their complex reaction mechanisms and wide variety of resulting products. Of particular interest is how these enzymes specify product outcome (2). Recent reports have demonstrated that specificity can be dramatically shifted by changes in a small number of amino acid resid...

A Single Residue Switch Converts Abietadiene Synthase into a Pimaradiene Specific Cyclase

“…In previous work, we found that a specific single residue change (Ile to Thr) was sufficient to "short circuit" the complex cyclization reaction catalyzed by ent-kaurene synthases to instead produce ent-pimara- 8(14),15-diene, presumably by deprotonation of a mechanistically relevant ent-pimar-15-en-8-yl + intermediate. 5 Here we demonstrate that AgAS product output can be switched from abietadienes to pimaradienes by a similar single residue change, albeit with subtle differences that have important mechanistic implications.…”

“…5 Alignment of these conifer diterpene cyclases with those from rice (Oryza satiVa) utilized in our previous study revealed that there was only a small change in aliphatic side chain size at the corresponding position. In particular, AgAS and PaAS contain a Val at this position, while PaPS has a Leu ( Figure 1).…”

“…The increased active site volume resulting from the Thr for Ile mutation in our previous study 5 left open the possibility that the introduced hydroxyl group might coordinate a novel active site water that directly deprotonated the relevant pimarenyl + intermediate. However, the Ser for Ala mutant reported here reinforces our hypothesis that the introduced hydroxyl group stabilizes the pimar-15-en-8-yl + intermediate, albeit with the necessary shift in active site position presumably dictated by the difference in CPP configuration.…”

16-Aza-ent-beyerane and 16-Aza-ent-trachylobane: Potent Mechanism-Based Inhibitors of Recombinant ent-Kaurene Synthase from Arabidopsis thaliana

“…11 An alternative path to the atiserane diterpenes would involve ring opening of a free or enzyme-bound trachylobane intermediate.…”

“…Kinetic runs were conducted at 10 nM protein concentration according to recently published procedures. 11 Reactions were initiated by addition of [15-3 H]ent-copalyl PP (5), 14e and [14-3 H]ent-kaurene was isolated by extraction and analyzed by liquid scintillation counting. 11,43 (Table 3) Studies with native KS partially purified from wild cucumber (Marah macrocarpus) seeds demonstrated that ring rearrangement occurs during the enzyme catalyzed cyclization of entcopalyl PP.…”