Linalyl Diphosphate Research Articles

Mutational Analysis of (+)-Limonene Synthase.

The monoterpene limonene is produced by the enzyme limonene synthase in one of the simplest terpene cyclization reactions. The enzyme can use linalyl diphosphate (LPP) and neryl diphosphate (NPP) as substrates in addition to the naturally occurring substrate geranyl diphosphate (GPP), but the relationship among the three alternative substrates is not well understood. We explored the (+)-limonene synthase ((+)-LS) reaction using site-directed mutagenesis with the three different substrates (GPP, NPP, and LPP) to tease out details of the mechanism. In total, 23 amino acid positions in the active site of (+)-LS were targeted for mutation. In all cases, substitution with Ala resulted in a significant loss of enzyme activity using GPP or NPP as the substrate, but the mutations fell into two groups depending on the effect of using LPP as a substrate: group 1 mutations resulted in the loss of activity with all three substrates (GPP, NPP, and LPP); group 2 mutations resulted in loss of activity with GPP and NPP, but retained near-WT activity with LPP as a substrate. Importantly, mutations resulting in loss of activity with LPP but retention of activity with GPP and NPP were never observed. These data, in combination with the substrate order of reactivity for the WT enzyme (LPP > NPP > GPP), are consistent with a role for LPP as an intermediate in the (+)-LS reaction using either GPP or NPP as a substrate.

Origin and functional differentiation of (E)-β-ocimene synthases reflect the expansion of monoterpenes in angiosperms.

The acquisition of new metabolic activities is a major force driving evolution. We explored, from the perspectives of gene family expansion and the evolutionary adaptability of proteins, how new functions have arisen in which terpene synthases diverged. Monoterpenoids are diverse natural compounds that can be divided into cyclic and acyclic skeleton forms according to their chemical structure. We demonstrate, through phylogenetic reconstructions and genome synteny analyses, that the (E)-β-ocimene synthases, which are acyclic monoterpene synthases (mTPSs), appear to have arisen several times in independent lineages during plant evolution. Bioinformatics analyses and classical mutation experiments identified four sites (I388, F420, S446, and F485) playing important roles in the neofunctionalization of mTPSs. Incubation of neryl diphosphate with Salvia officinalis 1,8-cineole synthase (SCS) and mutated proteins show that these four sites obstruct the isomerization of geranyl diphosphate. Quantum mechanical/molecular mechanical molecular dynamics simulations of models of SCS, SCSY420F/I446S, and SCSN338I/Y420F/I446S/L485F with (3R)-linalyl diphosphate suggest that mutations changed the configuration of the intermediate to obtain new activities. These results provide new perspectives on the evolution of mTPSs, explain the convergent evolution of (E)-β-ocimene synthases at the molecular level, and identify key residues to control the specificity of engineered mTPSs.

Direct Evidence of an Enzyme-Generated LPP Intermediate in (+)-Limonene Synthase Using a Fluorinated GPP Substrate Analog.

Linalyl diphosphate (LPP) is the postulated intermediate in the enzymatic cyclization of monoterpenes catalyzed by terpene synthases. LPP is considered an obligate intermediate due to the conformationally restrictive trans-C2-C3 double bond of the substrate, geranyl diphosphate (GPP), which precludes the proper positioning of carbons C1 and C6 to enable cyclization. However, because of the complexity of potential carbocation-mediated rearrangements in these enzymatic reactions, it has proven difficult to directly demonstrate the formation of LPP despite significant efforts. Here we synthesized a fluorinated substrate analog, 8,9-difluorogeranyl diphosphate (DFGPP), which is designed to allow initial ionization/isomerization and form the fluorinated equivalent of LPP (DFLPP) while preventing the subsequent ionization/cyclization to produce the α-terpinyl cation. Steady-state kinetic studies with the model enzyme (+)-limonene synthase (LS) under catalytic conditions show that the cyclization of DFGPP is completely blocked and a single linear product, difluoromyrcene, is produced. When crystals of apo-LS are soaked with DFGPP under conditions limiting turnover of the enzyme, we show, using X-ray crystallography, that DFLPP is produced in the enzyme active site and trapped in the crystals. Clear electron density is observed in the active site of the enzyme, but it cannot be appropriately fit with a model for the DFGPP substrate analog, whereas it can accommodate an extended conformation of DFLPP. This result supports the current model for monoterpene cyclization by providing direct evidence of LPP as an intermediate.

QM/MM free energy simulations of the reaction catalysed by (4S)-limonene synthase involving linalyl diphosphate (LPP) substrate

A large number of terpenoid natural products are known to exist in nature. Terpene synthases are pivotal enzymes for the biosynthesis of diverse terpenoid skelotons. Monoterpene synthases are one type of terpene synthases responsible for the production of several hundreds of natural monoterpenes based on a very limited pool of substrates. Therefore, understanding detailed catalytic mechanisms of those enzymes are important for understanding the product specificity of terpene synthases. In this study, we present a detailed mechanistic description of the biosynthesis of the (4S)-α-terpinyl carbocation from (3S)-linalyl diphosphate (LPP) catalysed by (4S)-limonene synthase (LS) using two-dimensional QM/MM free energy (2D-PMF) simulations. Our estimated free energy barrier is in a reasonable agreement with the corresponding experimental kinetic data. We also perform the one-dimensional QM/MM free energy (1D-PMF) simulations and show that His579 can act as a general base to deprotonate (4S)-α-terpinyl carbocation and to generate the limonene product.

A detailed view on 1,8-cineol biosynthesis by Streptomyces clavuligerus.

The stereochemical course of the cyclisation reaction catalysed by the bacterial 1,8-cineol synthase from Streptomyces clavuligerus was investigated using stereospecifically deuterated substrates. In contrast to the well investigated plant enzyme from Salvia officinalis, the reaction proceeds via (S)-linalyl diphosphate and the (S)-terpinyl cation, while the final cyclisation reaction is in both cases a syn addition, as could be shown by incubation of (2-13C)geranyl diphosphate in deuterium oxide.

Stereochemical mechanism of two sabinene hydrate synthases forming antipodal monoterpenes in thyme (Thymus vulgaris)

The essential oil of Thymus vulgaris consists of a complex blend of mono- and sesquiterpenes that provides the plant with its characteristic aromatic odor. Several chemotypes have been described for thyme. In this study, we identified two enzymes of the sabinene hydrate chemotype which are responsible for the biosynthesis of its major monoterpene alcohols, (1S,2R,4S)-(Z)-sabinene hydrate and (1S,2S,4R)-(E)-sabinene hydrate. Both TPS6 and TPS7 are multiproduct enzymes that formed 16 monoterpenes and thus cover almost the whole monoterpene spectrum of the chemotype. Although the product spectra of both enzymes are similar, they form opposing enantiomers of their chiral products. Incubation of the enzymes with the potential reaction intermediates revealed that the stereospecificity of TPS6 and TPS7 is determined by the formation of the first intermediate, linalyl diphosphate. Since TPS6 and TPS7 shared an amino acid sequence identity of 85%, a mutagenesis study was employed to identify the amino acids that determine the stereoselectivity. One amino acid position had a major influence on the stereochemistry of the formed products. Based on comparative models of TPS6 and TPS7 protein structures with the GPP substrate docked in the active site pocket, the influence of this amino acid residue on the reaction mechanism is discussed.

Inhibition of monoterpene cyclases by inert analogues of geranyl diphosphate and linalyl diphosphate

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which then cyclizes to the various monoterpene skeletons. X-ray crystal structures of these enzymes complexed with suitable analogues of the substrate and intermediate could provide a clearer view of this universal, but cryptic, step of monoterpenoid cyclase catalysis. Toward this end, the functionally inert analogues 2-fluorogeranyl diphosphate, (±)-2-fluorolinalyl diphosphate, and (3R)- and (3S)-homolinalyl diphosphates (2,6-dimethyl-2-vinyl-5-heptenyl diphosphates) were prepared, and compared to the previously described substrate analogue 3-azageranyl diphosphate (3-aza-2,3-dihydrogeranyl diphosphate) as inhibitors and potential crystallization aids with two representative monoterpenoid cyclases, (–)-limonene synthase and (+)-bornyl diphosphate synthase. Although these enantioselective synthases readily distinguished between (3R)- and (3S)-homolinalyl diphosphates, both of which were more effective inhibitors than was 3-azageranyl diphosphate, the fluorinated analogues proved to be the most potent competitive inhibitors and have recently yielded informative liganded structures with limonene synthase.

Alternative termination chemistries utilized by monoterpene cyclases: chimeric analysis of bornyl diphosphate, 1,8-cineole, and sabinene synthases

Monoterpene cyclization reactions are initiated by ionization and isomerization of geranyl diphosphate, and proceed, via cyclization of bound linalyl diphosphate, through a series of carbocation intermediates with ultimate termination of the multistep cascade by deprotonation or nucleophile capture. Three structurally and mechanistically related monoterpene cyclases from Salvia officinalis, (+)-sabinene synthase (deprotonation to olefin), 1,8-cineole synthase (water capture), and (+)-bornyl diphosphate synthase (diphosphate capture), were employed to explore the structural determinants of these alternative termination chemistries. Results with chimeric recombinant enzymes, constructed by reciprocally substituting regions of sabinene synthase with the corresponding sequences from bornyl diphosphate synthase or 1,8-cineole synthase, demonstrated that exchange of the C-terminal catalytic domain is sufficient to completely switch the resulting product profile. Exchange of smaller sequence elements identified a region of roughly 70 residues from 1,8-cineole synthase that, when substituted into sabinene synthase, conferred the ability to produce 1,8-cineole. A similar strategy identified a small region of bornyl diphosphate synthase important in conducting the anti-Markovnikov addition to the bornane skeleton. Observations made with these chimeric monoterpene cyclases are discussed in the context of the recently determined crystal structure for bornyl diphosphate synthase.

Mechanism of monoterpene cyclization: stereochemistry of the transformation of noncyclizable substrate analogs by recombinant (−)-limonene synthase, (+)-bornyl diphosphate synthase, and (−)-pinene synthase

All monoterpene cyclases investigated to date are capable of overcoming the topological impediment to direct cyclization of the universal, acyclic C10 intermediate of isoprenoid biosynthesis geranyl diphosphate. Although strong suggestive evidence has been accumulated for the intermediary linalyl diphosphate in cyclase catalysis, all previous efforts to directly observe this product at the mandatory isomerization step have failed. (−)-4S-Limonene synthase from spearmint (Mentha spicata), (+)-bornyl diphosphate synthase from sage (Salvia officinalis), and (−)-pinene synthase from grand fir (Abies grandis) have been expressed in Escherichia coli and the recombinant enzymes have been isolated and purified. These enzymes were examined with the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and 2,3-methanogeranyl diphosphate to gain insight into the normally cryptic isomerization step of the reaction sequence. The analogs were catalytically active, affording acyclic olefins and alcohols as products. Chiral phase gas chromatography and mass spectrometry analysis provided evidence that the normal cyclization of geranyl diphosphate by (−)-4S-limonene synthase and by (−)-pinene synthase proceeds via preliminary isomerization to the bound tertiary intermediate 3S-linalyl diphosphate, whereas the cyclization catalyzed by (+)-bornyl diphosphate synthase proceeds via the intermediate 3R-linalyl diphosphate.

Molecular cloning and characterization of a new linalool synthase

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (−)-3 R-linalool and its acetate ester. A cloning strategy based upon the assumption that the responsible monoterpene synthase would resemble, in sequence, monoterpene cyclases from this plant family yielded a cDNA encoding the (−)-3 R-linalool synthase. The nucleotide sequence of this monoterpene synthase is similar to those of several monoterpene cyclases from the mint (Lamiaceae) family (62–72% identity), but differs substantially from that of 3 S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3 R-linalool synthase from Artemisia (52% identity; the functional role of this gene is uncertain). Heterologous expression in Escherichia coli of a truncated version of the cDNA (in which the plastidial transit peptide was deleted) allowed purification and characterization of the enzyme, which was shown to possess most properties similar to other known monoterpene cyclases, but with a K m value for the natural substrate, geranyl diphosphate, of 56 μM with k cat of 0.83 s −1. These kinetic constants for this 3 R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3 S-linalool synthase from Clarkia. Although linalyl diphosphate is an enzyme-bound intermediate of monoterpene cyclase reactions, this tertiary allylic isomer of the geranyl substrate is not an efficient precursor of linalool with the M. citrata synthase. Modeling of the active site of this linalool synthase from Mentha and comparison to the modeled active sites of phylogenetically related monoterpene cyclases revealed structural differences in the binding of the diphosphate moiety which initiates the ionization step of the electrophilic reaction sequence and in the access of water to the active site to permit stereoselective quenching of the initially formed carbocationic intermediate to produce 3 R-linalool.

Biosynthesis of Marine Natural Products: Isolation and Characterization of a Myrcene Synthase from Cultured Tissues of the Marine Red Alga Ochtodes secundiramea

The acyclic monoterpene myrcene is the likely progenitor of the unusual cytotoxic halogenated monoterpenes that are found in marine algae and that function as feeding deterrents to herbivores. Myrcene synthase was isolated from suspension cultures of the marine red alga Ochtodes secundiramea, representing the first enzyme of this type from a marine organism. The algal myrcene synthase produces exclusively myrcene from the natural substrate geranyl diphosphate (GDP), utilizes Mg+2 as the required divalent metal ion cofactor, has a molecular mass of about 69 kDa, and exhibits a pH optimum near 7.2. These features are similar to those of monoterpene synthases from terrestrial organisms. When incubated with neryl diphosphate (the cis-isomer of GDP), the O. secundiramea myrcene synthase produces the cyclic monoterpene limonene, whereas incubation with (±)linalyl diphosphate (the tertiary allylic isomer of geranyl diphosphate) yields both acyclic and cyclic monoterpenes. These results suggest that the enzyme is incapable of isomerizing geranyl diphosphate to linalyl diphosphate, a feature common to all monoterpene cyclases from terrestrial sources. The limited catalytic capability of the myrcene synthase may reflect the ancient evolutionary origin of the producing organism. The ability to assay this enzyme in cultured algae, grown under strictly defined conditions, provides an unparalleled opportunity to delineate factors eliciting the biosynthesis of this class of secondary metabolites, to investigate the metabolic pathway leading to the halogenated monoterpenes, and to determine their role in the chemical ecology of marine algae.

Mechanism of Monoterpene Cyclization: Stereochemical Aspects of the Transformation of Noncyclizable Substrate Analogs by Recombinant (−)-Limonene Synthase, (+)-Bornyl Diphosphate Synthase, and (−)-Pinene Synthase

The tightly coupled nature of the reaction sequence catalyzed by monoterpene synthases has prevented direct observation of the topologically required isomerization step leading from geranyl diphosphate to the presumptive, enzyme-bound, tertiary allylic intermediate linalyl diphosphate, which ultimately cyclizes to the various monoterpene skeletons. Previous experimental approaches using the noncyclizable substrate analogs 6,7-dihydrogeranyl diphosphate and racemic methanogeranyl diphosphate, in attempts to dissect the cryptic isomerization step from the normally coupled reaction sequence, were thwarted by the limited product available from native monoterpene synthases and by the inability to resolve chiral monoterpene products at the microscale. These approaches were revisited using three recombinant monoterpene synthases and chiral phase capillary gas chromatographic methods to separate antipodal products of the substrate analogs. The recombinant monoterpene olefin synthases, (−)-limonene synthase from spearmint and (−)-pinene synthase from grand fir, yielded essentially only achiral, olefin products (corresponding to the respective analogs and homologs of myrcene, trans-ocimene and cis-ocimene) from 6,7-dihydrogeranyl diphosphate and (2S,3R)-methanogeranyl diphosphate; no significant amounts of terpenols or homoterpenols were formed, nor was direct evidence obtained for the formation of the anticipated analog and homolog of the tertiary intermediate linalyl diphosphate (i.e., 6,7-dihydrolinalyl diphosphate and homolinalyl diphosphate, respectively). In the case of recombinant (+)-bornyl diphosphate synthase from common sage, the achiral olefins were generated, as before, from 6,7-dihydrogeranyl diphosphate and (2R,3S)-methanogeranyl diphosphate, but 6,7-dihydrolinalool and homolinalool also comprised significant components of the respective product mixtures, indicating greater access of water to the active site of this enzyme compared to the olefin synthases; again, no direct evidence for the production of 6,7-dihydrolinalyl diphosphate or homolinalyl diphosphate was obtained. Resolution of the terpenol products of (+)-bornyl diphosphate synthase, by chiral phase separation, revealed the predominant formation of (3R)-dihydrolinalool from dihydrogeranyl diphosphate and of (4S)-homolinalool from (2R,3S)-methanogeranyl diphosphate. The opposite stereochemistries of these products indicates water trapping from opposite faces of the corresponding tertiary carbocationic intermediates of the respective reactions, a phenomenon that appears to result from the binding conformations of these substrate analogs. Although these experiments failed to provide direct evidence for the tertiary intermediate of the tightly coupled isomerization-cyclization sequence, they did reveal a mechanistic difference between the olefin synthases and bornyl diphosphate synthase involving access of water as a participant in the reaction.

CHARACTERIZATION OF A MYRCENE SYNTHASE FROM SUSPENSION CULTURES OF THE MARINE RED ALGA OCHTODES SECUNDIRAMEA

Wise, M. L.1, Rorrer, G. L.2, Polzin, J. J.2, Croteau, R. B.1 1Institute of Biological Chemistry, Washington State University, Pullman, WA. 99164 USA; 2 Department of Chemical Engineering, Oregon State University, Corvallis, OR. 96331USAA monoterpene synthase from suspension cultures of the marine red alga Ochtodes secundiramea is shown to biosynthesize myrcene from geranyl diphosphate (GPP) using cell free extracts. This is the first in vitro characterization of a monoterpene synthase from a marine organism. Myrcene is the likely progenitor of the unusual halogenated monoterpenes characteristic of this marine alga and, as such, represents a key step in the biosynthetic pathway. Based on mechanistic considerations from reaction with the biologically relevant substrate GPP, as well as neryl diphosphate (the cis isomer of GPP) and linalyl diphosphate (LPP), the enzyme appears incapable of catalyzing the isomerization of GPP to LPP, a mechanistic feature of most terrestrial monoterpene synthases, perhaps reflecting its evolutionarily ancient origin. The ability to assay and quantitatively monitor the expression of this enzyme in suspension cultures, under strictly defined growth conditions, presents an unparalleled opportunity to delineate, at the molecular level, factors eliciting the biosynthesis of this class of secondary metabolites, to evaluate the metabolic pathway leading to halogenated monoterpenes and to investigate their role in the chemical ecology of marine algae.

Studies on the Biosynthesis of the Antibiotic Moenomycin A

Feeding experiments ( 13C and 15N-labeled precursors) shed light on the biosynthetic origin of the chromophore (unit A of 1), the N-acetyl groups, the 4-C-methyl group of the moenuronamide unit (part F of 1), the sugar units, and the lipid part (unit I of 1) of the antibiotic moenomycin A(1). The lipid part is completely isoprenoid and is constructed via the non-mevalonate pathway. The central C 10 part originates from a precursor like geranyl or linalyl diphosphate and is formed by a route involving ring formation between C-2 and C-6 of the monoterpene unit, two successive rearrangements to give a 7-membered ring intermediate and cleavage of the ring between C-5 and C-11 (moenocinol numbering).

On the biosynthesis of moenocinol, the lipid part of the moenomycin antibiotics

The lipid part of the moenomycins is completely isoprenoid and is constructed via the non-mevalonate pathway. The central C 10 part originates from a precursor like geranyl or linalyl diphosphate and is formed by a route involving ring formation between C-2 and C-6 of the monoterpene unit, two successive rearrangements to give a seven-membered ring intermediate and cleavage of the ring at the C-5 and C-11 bond (moenocinol numbering).

Hydrolysis of isoprenyl diphosphates with the acid phosphatase fromCinnamomum camphora

Direct observations of the enzymatic hydrolysis of C10 acyclic allylic isoprenyl diphosphates by an acid phosphatase from the leaves ofCinnamomum camphora (camphor tree) were made using1H and31P NMR spectrometers. The measurements indicated that the allylic primary diphosphates, geranyl diphosphate and neryl diphosphate, were hydrolyzed to their corresponding alcohols in a sequential manner via their corresponding monophosphates, whereas the allylic tertiary diphosphate, linalyl diphosphate, was hydrolyzed only to its corresponding monophosphate.

Biosynthesis of Cyclic Monoterpenoids. Involvement of Stereochemically Specified Linalyl Cation in the Cyclization of C10-Prenyl Chain

Abstract Incubation of geranyl diphosphate (GPP) and the enantiomers and racemate of linalyl diphosphate (LPP) with the limonene synthetase isolated from Mentha spicata resulted in the conversion of GPP and (S)-LPP into (S)-limonene; this can be taken as evidence that linalyl cation having not only the (S)-LPP-like configuration but also C-1 and C-6 double bonds situated in the anti spatial arrangement is involved in the process of the cyclization of GPP to (S)-limonene. The intermediacy of the linalyl cation was supported by the evaluation of the potential energy of the cation with the MO calculation.

Hydrolysis of bismetallic complexes of linalyldiphosphate and their participation in the biosynthesis of cyclic monoterpenes

The allylic diphosphate ester linalyldiphosphate forms mono- and bis-metallic complexes with Mg2+ and Mn2+. Complexes of the trianion of the ligand are 50 to 100 times more stable than those of the dianion. Mg2+ and Mn2+ are bound in the monometallic complex 103 and 104 times more strongly than in the bismetallic species.The rate constant of the uncatalysed hydrolysis of linalyldiphosphate at pH 7.0 is 1.2 × 104s–1, which is higher than reported values for the primary diphosphates. Mg2+ and Mn2+ affect its rate of hydrolysis and it is shown that the rate enhancement observed is a function of the concentration of the bismetallic complexes.The rate of cyclic terpene hydrocarbon biosynthesis catalysed by carbocyclase from Citrus limonum also correlates with the concentration of the bismetallic complexes.Linalyl monophosphate, which is not a substrate for carbocyclase hydrolyses at a rate proportional to the concentration of the monometallic complex of Mn2+. The neutral charge of this complex, as opposed to the positively charged bismetallic complex of linalyl diphosphate may be an explanation of the absence of utilization of this complex as a substrate.It appears from the data presented and from previous evidence that bismetallic complexes of allylic diphosphates are the most reactive species in their hydrolysis and the only reactive species in the enzymic cyclization of these precursors of monoterpenes.