Functional and Structural Characterization of a (+)-Limonene Synthase from <i>Citrus sinensis</i>

Morehouse, B.R.; Kumar, Rajeev; Matos, Jason O.; Olsen, Sarah Naomi; Entova, Sonya; Oprian, Daniel D.

doi:10.1021/acs.biochem.7b00143

Cited by 48 publications

(70 citation statements)

References 40 publications

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“…Oprian and colleagues suggest that it might help block water from entering the active site, stabilize reaction intermediates, and/or stabilize metal ion binding through hydrogen bonding to an aspartate metal ligand. 194 …”

Section: Class I Terpenoid Cyclasesmentioning

confidence: 99%

“…Despite the dramatically different substrate binding conformations required for catalysis, it is striking that the amino acid residues lining the active sites of (+)-limonene synthase and (−)-limonene synthase are highly conserved ( Figure 39 ). 194 What, then, provides the stereochemical imperative for substrate binding and catalysis?…”

Section: Class I Terpenoid Cyclasesmentioning

confidence: 99%

“… (A) Comparison of (+)-limonene synthase (blue) and (−)-limonene synthase (green) showing that most active site residues are conserved. Reproduced from ref ( 194 ). Copyright 2017 American Chemical Society.…”

Section: Class I Terpenoid Cyclasesmentioning

confidence: 99%

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Structural and Chemical Biology of Terpenoid Cyclases

2017

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The year 2017 marks the twentieth anniversary of terpenoid cyclase structural biology: a trio of terpenoid cyclase structures reported together in 1997 were the first to set the foundation for understanding the enzymes largely responsible for the exquisite chemodiversity of more than 80000 terpenoid natural products. Terpenoid cyclases catalyze the most complex chemical reactions in biology, in that more than half of the substrate carbon atoms undergo changes in bonding and hybridization during a single enzyme-catalyzed cyclization reaction. The past two decades have witnessed structural, functional, and computational studies illuminating the modes of substrate activation that initiate the cyclization cascade, the management and manipulation of high-energy carbocation intermediates that propagate the cyclization cascade, and the chemical strategies that terminate the cyclization cascade. The role of the terpenoid cyclase as a template for catalysis is paramount to its function, and protein engineering can be used to reprogram the cyclization cascade to generate alternative and commercially important products. Here, I review key advances in terpenoid cyclase structural and chemical biology, focusing mainly on terpenoid cyclases and related prenyltransferases for which X-ray crystal structures have informed and advanced our understanding of enzyme structure and function.

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Section: Class I Terpenoid Cyclasesmentioning

confidence: 99%

Section: Class I Terpenoid Cyclasesmentioning

confidence: 99%

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Structural and Chemical Biology of Terpenoid Cyclases

2017

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“…caryolan-1-ol HO as previously described for substrates and substrate analogs of the enzyme (+)-limonene synthase. 21,46,47 Technologies (New Jersey). The genes were in a pET-28a (+) vector as a NcoI (5'-end) and…”

Section: Discussionmentioning

confidence: 99%

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Matos

Kumar

Alison

et al. 2020

Preprint

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Accession CodesThe atomic coordinates and structure factors for the DFFPP-PS complex have been deposited in the Protein Data Bank. RCSB PDB entry 6WKC (WT-Mg), 6WKD (WT-DFFPP), 6WKE DFFPP) for the respective models. ABBREVIATIONS FPP, farnesyl diphosphate; DFFPP, 12,13-difluorofarnesyl diphosphate; PS, pentalenene synthase, CS, caryolan-1-ol synthase. SUPPORTING INFORMATION AVAILABLE AbstractMost terpene synthase reactions follow Markovnikov rules for formation of high energy carbenium ion intermediates. However, there are notable exceptions. For example, pentalenene synthase (PS) undergoes an initial anti-Markovnikov cyclization reaction followed by a 1,2hydride shift to form an intermediate humulyl cation with positive charge on the secondary carbon C9 of the farnesyl diphosphate substrate. The mechanism by which these enzymes stabilize and guide regioselectivity of secondary carbocations has not heretofore been elucidated.In an effort to better understand these reactions, we grew crystals of apo-PS, soaked them with the non-reactive substrate analog 12,13-difluorofarnesyl diphosphate, and solved the x-ray structure of the resulting complex at 2.2 Å resolution. The most striking feature of the active site structure is that C9 is positioned 3.5 Å above the center of the side chain benzene ring of residue F76, perfectly poised for stabilization of the charge through a cation-p interaction. In addition, the main chain carbonyl of I177 and neighboring intramolecular C6,C7-double bond are positioned to stabilize the carbocation by interaction with the face opposite that of F76.Mutagenesis experiments also support a role for residue 76 in cation-p interactions. Most interesting is the F76W mutant which gives a mixture of products that likely result from stabilizing a positive charge on the adjacent secondary carbon C10 in addition to C9 as in the wild-type enzyme. The crystal structure of the F76W mutant clearly shows carbons C9 and C10 centered above the fused benzene and pyrrole rings of the indole side chain, respectively, such that a carbocation at either position could be stabilized in this complex, and two anti-Markovnikov products, pentalenene and humulene, are formed. Finally, we show that there is a rough correlation (although not absolute) of an aromatic side chain (F or Y) at position 76 in related terpene synthases from Streptomyces that catalyze similar anti-Markovnikov addition reactions.

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“…Because of a fairly high boiling point (176 °C) and high standard enthalpy of combustion (− 6100 kJ/mol), limonene represents a promising highdensity isoprenoid-derived biofuel [267]. The biosynthesis of limonene requires two biosynthetic enzymes for the formation of GPP by GPPS and cyclization of GPP into limonene catalyzed by LMS [280]. Several attempts have been made to engineer limonene production in plants via overexpression of 4S-LMS (Fig.…”

Section: R-limonene (C 10 H 16 Monoterpene)mentioning

confidence: 99%

Strategies for the production of biochemicals in bioenergy crops

Lin

Eudes

2020

Biotechnol Biofuels

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Industrial crops are grown to produce goods for manufacturing. Rather than food and feed, they supply raw materials for making biofuels, pharmaceuticals, and specialty chemicals, as well as feedstocks for fabricating fiber, biopolymer, and construction materials. Therefore, such crops offer the potential to reduce our dependency on petrochemicals that currently serve as building blocks for manufacturing the majority of our industrial and consumer products. In this review, we are providing examples of metabolites synthesized in plants that can be used as bio-based platform chemicals for partial replacement of their petroleum-derived counterparts. Plant metabolic engineering approaches aiming at increasing the content of these metabolites in biomass are presented. In particular, we emphasize on recent advances in the manipulation of the shikimate and isoprenoid biosynthetic pathways, both of which being the source of multiple valuable compounds. Implementing and optimizing engineered metabolic pathways for accumulation of coproducts in bioenergy crops may represent a valuable option for enhancing the commercial value of biomass and attaining sustainable lignocellulosic biorefineries.

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Functional and Structural Characterization of a (+)-Limonene Synthase from Citrus sinensis

Cited by 48 publications

References 40 publications

Structural and Chemical Biology of Terpenoid Cyclases

Structural and Chemical Biology of Terpenoid Cyclases

Mechanism Underlying Anti-Markovnikov Addition in the Reaction of Pentalenene Synthase

Strategies for the production of biochemicals in bioenergy crops

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