Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases

O’Maille, Paul E.; Malone, Arthur; Dellas, Nikki; Hess, B. Andes; Smentek, Lidia; Sheehan, Iseult; Greenhagen, Bryan T.; Chappell, Joe; Manning, Gerard; Noel, Joseph P.

doi:10.1038/nchembio.113

Cited by 188 publications

(196 citation statements)

References 34 publications

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“…A growing body of evidence suggests that many enzymes exhibit broad catalytic specificity and/or promiscuity 11,17 , particularly enzymes in secondary metabolism [44][45][46][47][48] . Broad specificity may therefore relate to the recent divergence of secondary and specialized metabolism enzymes relative to primary metabolic enzymes, as well as the intensity of selection being weaker than it is for enzymes in primary metabolism 45,49 .…”

Section: Discussionmentioning

confidence: 99%

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

et al. 2012

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Optimization processes, such as evolution, are constrained by diminishing returns-the closer the optimum, the smaller the benefit per mutation, and by tradeoffs-improvement of one property at the cost of others. However, the magnitude and molecular basis of these parameters, and their effect on evolutionary transitions, remain unknown. Here we pursue a complete functional transition of an enzyme with a 410 9 -fold change in the enzyme's selectivity using laboratory evolution. We observed strong diminishing returns, with the initial mutations conferring 425-fold higher improvements than later ones, and asymmetric tradeoffs whereby the gain/loss ratio of the new/old activity decreased 400-fold from the beginning of the trajectory to its end. We describe the molecular basis for these phenomena and suggest they have an important role in shaping natural proteins. These findings also suggest that the catalytic efficiency and specificity of many natural enzymes may be far from their optimum.

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Section: Discussionmentioning

confidence: 99%

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

et al. 2012

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“…However, owing to descent with modification and its historical record encoded in genomes, recent technological advances have facilitated the generation of large-scale ancestral mutant libraries based on phylogenies of extant enzyme sequences (Weinreich et al 2006;O'Maille et al 2008;Goldsmith and Tawfik 2012). These developments make it possible to peer into the past with an admittedly slightly out of focus "telescope."…”

Section: Future Perspectivesmentioning

confidence: 99%

“…TPSs and type III PKSs often produce one major product together with a number of minor products. Moreover, the product distribution profiles of these enzymes are often conditionally sensitive and are exquisitely attuned to minor mutational changes within and around the active site (Austin et al 2004;O'Maille et al 2008). Although TPSs and PKSs belong to disparate protein folds, they converge to exploit similar catalytic principles to direct cyclization of their respective highly reactive intermediates.…”

Section: The Role Of Catalytic Promiscuity In Enzyme Evolvabilitymentioning

confidence: 99%

The Remarkable Pliability and Promiscuity of Specialized Metabolism

Weng

Noel

2012

Cold Spring Harbor Symposia on Quantitative Biology

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Metabolic pathways are often considered "perfected" or at least predictable as substrates efficiently rearrange into products through the intervention of an optimized enzyme. Moreover, single catalytic steps link up, forming a myriad of metabolic circuits that are often modeled with a high degree of certainty. However, on closer examination, most enzymes are not precise with respect to their activity, using not just one substrate but often a variety and producing not just one product but a diversity. Hence, the metabolic systems assembled from enzymes possessing varying degrees of what can be termed catalytic promiscuity are not clear-cut and restrictive; rather, they may at times operate stochastically in the intracellular milieu. This "messiness" complicates our understanding of normal and aberrant cellular behavior, while paradoxically sowing the seeds for future advantageous metabolic adaptations for host organisms. Catalytic promiscuity is intrinsically associated with the dynamic nature of enzyme structures and their chemical mechanisms, both key to enzyme and metabolic evolvability. In addition to primary (core) metabolism, which is essential for survival, organisms also possess highly elaborated secondary (specialized) metabolic systems. These specialized enzymes and pathways often provide unique adaptive strategies for a myriad of organisms and their populations in challenging and changing ecosystems. Generally, enzymes of specialized metabolism show attenuated kinetic activities and expanded catalytic promiscuity compared with their phylogenetic relatives rooted in primary metabolism. We propose that evolvability may be a selected trait in many specialized metabolic systems spread across populations of organisms exposed to continually fluctuating biotic and abiotic environmental pressures. As minor metabolites arising from catalytic messiness provided enhanced population fitness, specificity relaxed, and catalytic efficiency was attenuated. This updated view provides a mechanistic basis for reaching a deeper understanding of the evolutionary underpinnings of the explosion of chemodiversity in nature.Fundamentally, metabolism is a chemical property of living organisms, defined as the collection of enzymecatalyzed chemical reactions that synthesize or deconstruct metabolic chemicals necessary to sustain life and contribute to the reproductive success of host populations in the face of ever-changing environmental pressures. Similarly to other catalysts, enzymes do not alter the equilibrium of the chemical reactions but enhance the rate of the reactions by lowering their activation energies (Cook and Cleland 2007). Reactions catalyzed by enzymes are generally precise and efficient, involving specific metabolic substrates transformed into predictable metabolic products using defined catalytic mechanisms. This ordered view of one enzyme -one mechanism -one product comes from seminal discoveries made by many investigators during the last century, focused for excellent practical reasons on the phylogenetically...

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“…By systematically exploring the relationship between sequence and function, Yoshikuni et al (2006) identified and mutated a set of hot spots near the active site of sesquiterpene synthases that results in biocatalysts selectively catalyzing the formation of a spectrum of sesquiterpene products). Furthermore, O'Maille et al (2008) systematically investigated the relationship between a combination of nine naturally occurring amino acid substitutions and the resulting sesquiterpene product spectra. However, the application of the resulting catalysts was not attempted in planta for diversifying plant metabolism.…”

Section: Online)mentioning

confidence: 99%

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

et al. 2012

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Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.

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Quantitative exploration of the catalytic landscape separating divergent plant sesquiterpene synthases

Cited by 188 publications

References 34 publications

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

Diminishing returns and tradeoffs constrain the laboratory optimization of an enzyme

The Remarkable Pliability and Promiscuity of Specialized Metabolism

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

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