Unusual features of a recombinant apple alpha-farnesene synthase

A recombinant alpha-farnesene synthase from apple (Malus x domestica), expressed in Escherichia coli, showed features not previously reported. Activity was enhanced 5-fold by K(+) and all four isomers of alpha-farnesene, as well as beta-farnesene, were produced from an isomeric mixture of farnesyl diphosphate (FDP). Monoterpenes, linalool, (Z)- and (E)-beta-ocimene and beta-myrcene, were synthesised from geranyl diphosphate (GDP), but at 18% of the optimised rate for alpha-farnesene synthesis from FDP. Addition of K(+) reduced monoterpene synthase activity. The enzyme also produced alpha-farnesene by a reaction involving coupling of GDP and isoprenyl diphosphate but at <1% of the rate with FDP. Mutagenesis of active site aspartate residues removed sesquiterpene, monoterpene and prenyltransferase activities suggesting catalysis through the same active site. Phylogenetic analysis clusters this enzyme with isoprene synthases rather than with other sesquiterpene synthases, suggesting that it has evolved differently from other plant sesquiterpene synthases. This is the first demonstration of a sesquiterpene synthase possessing prenyltransferase activity.     

Rapid regulation of the methylerythritol 4-phosphate pathway during isoprene synthesis

More volatile organic carbon is lost from plants as isoprene than any other molecule. This flux of carbon to the atmosphere affects atmospheric chemistry and can serve as a substrate for ozone production in polluted air. Isoprene synthesis may help leaves cope with heatflecks and active oxygen species. Isoprene synthase, an enzyme related to monoterpene synthases, converts dimethylallyl diphosphate derived from the methylerythritol 4-phosphate pathway to isoprene. We used dideuterated deoxyxylulose (DOX-d(2)) to study the regulation of the isoprene biosynthetic pathway. Exogenous DOX-d(2) displaced endogenous sources of carbon for isoprene synthesis without increasing the overall rate of isoprene synthesis. However, at higher concentrations, DOX-d(2) completely suppressed isoprene synthesis from endogenous sources and increased the overall rate of isoprene synthesis. We interpret these results to indicate strong feedback control of deoxyxylulose-5-phosphate synthase. We related the emission of labeled isoprene to the concentration of labeled dimethylallyl diphosphate in order to estimate the in situ K(m) of isoprene synthase. The results confirm that isoprene synthase has a K(m) 10- to 100-fold higher for its allylic diphosphate substrate than related monoterpene synthases for geranyl diphosphate.     

Evolution of the isoprene biosynthetic pathway in kudzu

Isoprene synthase converts dimethylallyl diphosphate, derived from the methylerythritol 4-phosphate (MEP) pathway, to isoprene. Isoprene is made by some plants in substantial amounts, which affects atmospheric chemistry, while other plants make no isoprene. As part of our long-term study of isoprene synthesis, the genetics of the isoprene biosynthetic pathway of the isoprene emitter, kudzu (Pueraria montana), was compared with similar genes in Arabidopsis (Arabidopsis thaliana), which does not make isoprene. The MEP pathway genes in kudzu were similar to the corresponding Arabidopsis genes. Isoprene synthase genes of kudzu and aspen (Populus tremuloides) were cloned to compare their divergence with the divergence seen in MEP pathway genes. Phylogenetic analysis of the terpene synthase gene family indicated that isoprene synthases are either within the monoterpene synthase clade or sister to it. In Arabidopsis, the gene most similar to isoprene synthase is a myrcene/ocimene (acyclic monoterpenes) synthase. Two phenylalanine residues found exclusively in isoprene synthases make the active site smaller than other terpene synthase enzymes, possibly conferring specificity for the five-carbon substrate rather than precursors of the larger isoprenoids. Expression of the kudzu isoprene synthase gene in Arabidopsis caused Arabidopsis to emit isoprene, indicating that whether or not a plant emits isoprene depends on whether or not it has a terpene synthase capable of using dimethylallyl diphosphate.     

First isolation of an isoprene synthase gene from poplar and successful expression of the gene in Escherichia coli

For the first time, the complete functional gene for isoprene synthase has been isolated from poplar (Populus alba x Populus tremula). The gene was quite similar to known limonene and other monoterpene synthases, but was found to specifically catalyze the formation of isoprene from the precursor dimethylallyl diphosphate with only a marginal activity for the formation of the monoterpene limonene from geranyl diphosphate as compared with limonene synthases. Omitting the part of the gene that putatively encoded the signal peptide necessary for transport into the chloroplast led to an enhanced rate of isoprene formation by the recombinant protein.     

(E)-beta-ocimene and myrcene synthase genes of floral scent biosynthesis in snapdragon: function and expression of three terpene synthase genes of a new terpene synthase subfamily

Snapdragon flowers emit two monoterpene olefins, myrcene and (E)-beta-ocimene, derived from geranyl diphosphate, in addition to a major phenylpropanoid floral scent component, methylbenzoate. Emission of these monoterpenes is regulated developmentally and follows diurnal rhythms controlled by a circadian clock. Using a functional genomics approach, we have isolated and characterized three closely related cDNAs from a snapdragon petal-specific library that encode two myrcene synthases (ama1e20 and ama0c15) and an (E)-beta-ocimene synthase (ama0a23). Although the two myrcene synthases are almost identical (98%), except for the N-terminal 13 amino acids, and are catalytically active, yielding a single monoterpene product, myrcene, only ama0c15 is expressed at a high level in flowers and contributes to floral myrcene emission. (E)-beta-Ocimene synthase is highly similar to snapdragon myrcene synthases (92% amino acid identity) and produces predominantly (E)-beta-ocimene (97% of total monoterpene olefin product) with small amounts of (Z)-beta-ocimene and myrcene. These newly isolated snapdragon monoterpene synthases, together with Arabidopsis AtTPS14 (At1g61680), define a new subfamily of the terpene synthase (TPS) family designated the Tps-g group. Members of this new Tps-g group lack the RRx(8)W motif, which is a characteristic feature of the Tps-d and Tps-b monoterpene synthases, suggesting that the reaction mechanism of Tps-g monoterpene synthase product formation does not proceed via an RR-dependent isomerization of geranyl diphosphate to 3S-linalyl diphosphate, as shown previously for limonene cyclase. Analyses of tissue-specific, developmental, and rhythmic expression of these monoterpene synthase genes in snapdragon flowers revealed coordinated regulation of phenylpropanoid and isoprenoid scent production.     

Isolation and partial characterisation of a putative monoterpene synthase from Melaleuca alternifolia.

Melaleuca alternifolia (Cheel) is an Australia native tree harvested for its monoterpene-rich, essential oil. Monoterpene synthases (E.C. 4.2.3.20) were partially purified from the flush growth of the commercially important, high terpinen-4-ol chemotype of M. alternifolia. The purified fractions produced an acyclic monoterpene, linalool that is not present in the essential oil. To further characterise the monoterpene synthase, a cDNA library was constructed and 500 expressed sequence tags (ESTs) were sequenced to isolate putative terpene synthases. A single clone with similarity to the TspB gene sub-family of angiosperm monoterpene and isoprene synthases was isolated but was truncated at the 5' end. This single clone was used to design a probe for a cDNA library and was applied to isolate a full-length clone. This gene encoded a polypeptide 583 amino acids in length (67 kDa) including a putative transit peptide. Heterologous expression of the gene in Escherichia coli and subsequent assay of the recombinant enzyme did not result in the production of terpinen-4-ol, the major constituent of tea tree oil, or of its precursor sabinene hydrate. Significant quantities of linalool were observed in these assays, and in the assays of monoterpene synthase activity of a native enzyme in vitro, but the racemic nature of the linalool means that it may have a non-enzymatic origin.     

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.     

cDNA cloning, characterization, and functional expression of four new monoterpene synthase members of the Tpsd gene family from grand fir (Abies grandis).

Grand fir (Abies grandis) is a useful model system for studying the biochemistry, molecular genetics, and regulation of defensive oleoresin formation in conifers, a process involving both the constitutive accumulation of resin (pitch) in specialized secretory structures and the induced biosynthesis of monoterpenes and sesquiterpenes (turpentine) and diterpene resin acids (rosin) by nonspecialized cells at the site of injury. A similarity-based cloning strategy, employing primers designed to conserved regions of existing monoterpene synthases and anticipated to amplify a 1000-bp fragment, unexpectedly yielded a 300-bp fragment with sequence reminiscent of a terpenoid synthase. Utilization of this amplicon as a hybridization probe afforded four new, full-length cDNA species from a wounded fir stem cDNA library that appeared to encode four distinct monoterpene synthases. Expression in Escherichia coli, followed by enzyme assay with geranyl diphosphate (C(10)), farnesyl diphosphate (C(15)) and geranylgeranyl diphosphate (C(20)), and analysis of the terpene products by chiral phase gas chromatography and mass spectrometry confirmed that these sequences encoded four new monoterpene synthases, including (-)-camphene synthase, (-)-beta-phellandrene synthase, terpinolene synthase, and an enzyme that produces both (-)-limonene and (-)-alpha-pinene. The deduced amino acid sequences indicated these enzymes to be 618 to 637 residues in length (71 to 73 kDa) and to be translated as preproteins bearing an amino-terminal plastid targeting sequence of 50-60 residues. cDNA truncation to delete the transit peptide allowed functional expression of the "pseudomature" forms of these enzymes, which exhibited no change in product outcome as a result of truncation. Sequence comparison revealed that these new monoterpene synthases from grand fir are members of the Tpsd gene subfamily and resemble sesquiterpene (C(15)) synthases and diterpene (C(20)) synthases from conifers more closely than mechanistically related monoterpene synthases from angiosperm species. The availability of a nearly complete set of constitutive and inducible monoterpene synthases from grand fir (now numbering seven) will allow molecular dissection of the resin-based defense response in this conifer species, and detailed study of structure-function relationships among this large and diverse family of catalysts, all of which exploit the same stereochemistry in the coupled isomerization-cyclization reaction.     

Differential accumulation of dimethylallyl diphosphate in leaves and needles of isoprene- and methylbutenol-emitting and nonemitting species

The biosynthesis and emission of volatile plant terpenoids, such as isoprene and methylbutenol (MBO), depend on the chloroplastic production of dimethylallyl diphosphate (DMAPP). To date, it has been difficult to study the relationship of cellular DMAPP levels to emission of these volatiles because of the lack of a sensitive assay for DMAPP in plant tissues. Using a recent DMAPP assay developed in our laboratories, we report that species with the highest potential for isoprene and MBO production also exhibit elevated light-dependent DMAPP production, ranging from 110% to 1,063%. Even species that do not produce significant amounts of volatile terpenoids, however, exhibit some potential for light-dependent production of DMAPP. We used a nonaqueous fractionation technique to determine the intracellular distribution of DMAPP in isoprene-emitting cottonwood (Populus deltoides) leaves; approximately 65% to 70% of the DMAPP recovered at midday occurred in the chloroplasts, indicating that most of the light-dependent production of DMAPP was chloroplastic in origin. The midday concentration of chloroplastic DMAPP in cottonwood leaves is estimated to be 0.13 to 3.0 mM, which is consistent with the relatively high K(m)s that have been reported for isoprene synthases (0.5-8 mM). The results provide support for the hypothesis that the light dependence of isoprene and MBO emissions is in part due to controls over DMAPP production.     

Amino acid determinants of substrate selectivity in the Trypanosoma brucei sphingolipid synthase family

The substrate selectivity of four Trypanosoma brucei sphingolipid synthases was examined. TbSLS1, an inositol phosphorylceramide (IPC) synthase, and TbSLS4, a bifunctional sphingomyelin (SM)/ethanolamine phosphorylceramide (EPC) synthase, were inactivated by Ala substitutions of a conserved triad of residues His210, His253, and Asp257 thought to form part of the active site. TbSLS4 also catalyzed the reverse reaction, production of ceramide from sphingomyelin, but none of the Ala substitutions of the catalytic triad in TbSLS4 were able to do so. Site-directed mutagenesis identified residues proximal to the conserved triad that were responsible for the discrimination between charge and size of the different head groups. For discrimination between anionic (phosphoinositol) and zwitterionic (phosphocholine, phosphoethanolamine) head groups, doubly mutated V172D/S252F TbSLS1 and D172V/F252S TbSLS3 showed reciprocal conversion between IPC and bifunctional SM/EPC synthases. For differentiation of zwitterionic headgroup size, N170A TbSLS1 and A170N/N187D TbSLS4 showed reciprocal conversion between EPC and bifunctional SM/EPC synthases. These studies provide a mapping of the SLS active site and demonstrate that differences in catalytic specificity of the T. brucei enzyme family are controlled by natural variations in as few as three residue positions.     

Mutational analysis of a monoterpene synthase reaction: altered catalysis through directed mutagenesis of (-)-pinene synthase from Abies grandis

Two monoterpene synthases, (-)-pinene synthase and (-)-camphene synthase, from grand fir (Abies grandis) produce different product mixtures despite having highly homologous amino acid sequences and, presumably, very similar three-dimensional structures. The major product of (-)-camphene synthase, (-)-camphene, and the major products of (-)-pinene synthase, (-)-alpha-pinene, and (-)-beta-pinene, arise through distinct mechanistic variations of the electrophilic reaction cascade that is common to terpenoid synthases. Structural modeling followed by directed mutagenesis in (-)-pinene synthase was used to replace selected amino acid residues with the corresponding residues from (-)-camphene synthase in an effort to identify the amino acids responsible for the catalytic differences. This approach produced an enzyme in which more than half of the product was channeled through an alternative pathway. It was also shown that several (-)-pinene synthase to (-)-camphene synthase amino acid substitutions were necessary before catalysis was significantly altered. The data support a model in which the collective action of many key amino acids, located both in and distant from the active site pocket, regulate the course of the electrophilic reaction cascade.     

A non-synonymous nucleotide substitution can account for one evolutionary route to sesquiterpene synthase activity in the TPS-b subgroup

Plant sesquiterpene and hemiterpene synthases in the monoterpene synthase dominated TPS-b subgroup are thought to have evolved independently from a monoterpene synthase ancestor. A TPS-b sesquiterpene synthase from apple (MdAFS1), which predominantly produces α-farnesene, can also synthesize the monoterpene (E)-β-ocimene. The dual activity offered a functional link to an ancestral MdAFS1 enzyme and a rational basis for investigation of the evolution of TPS-b sesquiterpene enzymes. Protein modelling and mutagenesis analysis of the MdAFS1 active site identified a non-synonymous nucleotide substitution that could account for the requisite shift in substrate specificity necessary for the emergence of its sesquiterpene activity during the evolution of the TPS-b enzymes.     

异戊二烯合成酶研究进展

异戊二烯(Isoprene)的排放具有特殊的生物学功能,对大气环境具有重要影响,另外,异戊二烯也是一种具有广泛用途的化合物。在生物体内,异戊二烯是由异戊二烯合成酶(Isoprene synthase,Isps)催化烯丙基二磷酸(Dimethylallyl diphosphate,DMAPP)脱去焦磷酸(Pyrophosphate)而生成的。因此,作为异戊二烯合成过程中的关键酶,Isps在异戊二烯的自然排放和生物合成过程都发挥着重要的作用,对Isps的研究具有非常重要的意义。到目前为止,已经在多种植物中发现了该酶,研究表明,来源于不同生物的异戊二烯合成酶具有保守的结构特征和相似的生化性质。文中就Isps的最新研究进展进行综述,包括比较分析不同生物来源Isps的生化特征和结构特征,探讨催化机制,并对该酶在生物工程领域的应用进行展望。     

Molecular cloning and characterization of a new linalool synthase

Mentha citrata Ehrh. (bergamot mint; Lamiaceae) produces an essential oil containing only the acyclic monoterpenol (-)-3R-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 (--)-3R-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 3S-linalool synthase from Clarkia (41% identity; this composite gene appears to be of recent origin) and from that of 3R-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 microM with k(cat) of 0.83 s(-1). These kinetic constants for this 3R-linalool synthase are higher than those of any defined monoterpene cyclase, but the kinetic efficiency does not approach that reported for the 3S-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 3R-linalool.     

ISOPRENE SYNTHASE GENES FORM A MONOPHYLETIC CLADE OF ACYCLIC TERPENE SYNTHASES IN THE TPS‐B TERPENE SYNTHASE FAMILY

Many plants emit significant amounts of isoprene, which is hypothesized to help leaves tolerate short episodes of high temperature. Isoprene emission is found in all major groups of land plants including mosses, ferns, gymnosperms, and angiosperms; however, within these groups isoprene emission is variable. The patchy distribution of isoprene emission implies an evolutionary pattern characterized by many origins or many losses. To better understand the evolution of isoprene emission, we examine the phylogenetic relationships among isoprene synthase and monoterpene synthase genes in the angiosperms. In this study we identify nine new isoprene synthases within the rosid angiosperms. We also document the capacity of a myrcene synthase in Humulus lupulus to produce isoprene. Isoprene synthases and (E)‐β‐ocimene synthases form a monophyletic group within the Tps‐b clade of terpene synthases. No asterid genes fall within this clade. The chemistry of isoprene synthase and ocimene synthase is similar and likely affects the apparent relationships among Tps‐b enzymes. The chronology of rosid evolution suggests a Cretaceous origin followed by many losses of isoprene synthase over the course of evolutionary history. The phylogenetic pattern of Tps‐b genes indicates that isoprene emission from non‐rosid angiosperms likely arose independently.     

Transformation of acridone synthase to chalcone synthase.

Acridone synthase (ACS) and chalcone synthase (CHS) catalyse the pivotal reactions in the formation of acridone alkaloids or flavonoids. While acridone alkaloids are confined almost exclusively to the Rutaceae, flavonoids occur abundantly in all seed-bearing plants. ACSs and CHSs had been cloned from Ruta graveolens and shown to be closely related polyketide synthases which use N-methylanthraniloyl-CoA and 4-coumaroyl-CoA, respectively, as the starter substrate to produce the acridone or naringenin chalcone. As proposed for the related 2-pyrone synthase from Gerbera, the differential substrate specificities of ACS and CHS might be attributed to the relative volume of the active site cavities. The primary sequences as well as the immunological cross reactivities and molecular modeling studies suggested an almost identical spatial structure for ACS and CHS. Based on the Ruta ACS2 model the residues Ser132, Ala133 and Val265 were assumed to play a critical role in substrate specificity. Exchange of a single amino acid (Val265Phe) reduced the catalytic activity by about 75% but grossly shifted the specificity towards CHS activity, and site-directed mutagenesis replacing all three residues by the corresponding amino acids present in CHS (Ser132Thr, Ala133Ser and Val265Phe) fully transformed the enzyme to a functional CHS with comparatively marginal ACS activity. The results suggested that ACS divergently has evolved from CHS by very few amino acid exchanges, and it remains to be established why this route of functional diversity has developed in the Rutaceae only.     

Alteration of product formation by directed mutagenesis and truncation of the multiple-product sesquiterpene synthases delta-selinene synthase and gamma-humulene synthase

Two recombinant sesquiterpene synthases from grand fir, delta-selinene synthase and gamma-humulene synthase, each produce more than 30 sesquiterpene olefins from the acyclic precursor farnesyl diphosphate. These enzymes contain a pair of DDxxD motifs, on opposite lips of the presumptive active site, which are thought to be involved in substrate binding and could promote multiple orientations of the substrate alkyl chain from which multiple families of cyclic olefins could derive. Mutagenesis of the first aspartate of either DDxxD motif resulted in depressed k(cat), with lesser effect on K(m), for delta-selinene synthase and afforded a much simpler product spectrum composed largely of monocyclic olefins. Identical alterations in gamma-humulene synthase produced similar kinetic effects with a simplified product spectrum of mostly acyclic and monocyclic olefins. Although impaired in product diversity, none of the mutant synthases lost entirely the capacity to generate complex structures. These results confirm the catalytic significance of the DDxxD motifs and imply that they also influence permitted modes of cyclization. Deletion of an N-terminal arginine pair in delta-selinene synthase (an element potentially involved in substrate isomerization) altered kinetics without substantially altering product outcome. Finally, mutation of an active-site tyrosine residue thought to play a role in proton exchange had little influence; however, substitution of a nearby active site aspartate dramatically altered kinetics and product outcome.     

The transition-like state and Pi entrance into the catalytic a subunit of the biological engine A-ATP synthase

Archaeal A-ATP synthases catalyze the formation of the energy currency ATP. The chemical mechanisms of ATP synthesis in A-ATP synthases are unknown. We have determined the crystal structure of a transition-like state of the vanadate-bound form of catalytic subunit A (A_Vi) of the A-ATP synthase from Pyrococcus horikoshii OT3. Two orthovanadate molecules were observed in the A_Vi structure, one of which interacts with the phosphate binding loop through residue S238. The second vanadate is positioned in the transient binding site, implicating for the first time the pathway for phosphate entry to the catalytic site. Moreover, since residues K240 and T241 are proposed to be essential for catalysis, the mutant structures of K240A and T241A were also determined. The results demonstrate the importance of these two residues for transition-state stabilization. The structures presented shed light on the diversity of catalytic mechanisms used by the biological motors A- and F-ATP synthases and eukaryotic V-ATPases.     

Subunit interactions and glutamine utilization by Escherichia coli imidazole glycerol phosphate synthase

A selection strategy has been developed to identify amino acid residues involved in subunit interactions that coordinate the two half-reactions catalyzed by glutamine amidotransferases. The protein structures known for this class of enzymes have revealed that ammonia is shuttled over long distances and that each amidotransferase evolved different molecular tunnels for this purpose. The heterodimeric Escherichia coli imidazole glycerol phosphate (IGP) synthase was probed to assess if residues in the substrate amination subunit (HisF) are critical for the glutaminase activity in the HisH subunit. The activity of the HisH subunit is dependent upon binding of the nucleotide substrate at the HisF active site. This regulatory function has been exploited as a biochemical selection of mutant HisF subunits that retain full activity with ammonia as a substrate but, when constituted as a holoenzyme with wild-type HisH, impair the glutamine-dependent activity of IGP synthase. The steady-state kinetic constants for these IGP synthases with HisF alleles showed three distinct effects depending upon the site of mutation. For example, mutation of the R5 residue has similar effects on the glutamine-dependent amidotransfer reaction; however, k(cat)/K(m) for the glutaminase half-reaction was increased 10-fold over that for the wild-type enzyme with nucleotide substrate. This site appears essential for coupling of the glutamine hydrolysis and ammonia transfer steps and is the first example of a site remote to the catalytic triad that modulates the process. The results are discussed in the context of recent X-ray crystal structures of glutamine amidotransferases that relate the glutamine binding and acceptor binding sites.     

Biochemical Characterization of Stromal and Thylakoid-Bound Isoforms of Isoprene Synthase in Willow Leaves

Isoprene synthase is the enzyme responsible for the foliar emission of the hydrocarbon isoprene (2-methyl-1,3-butadiene) from many C₃ plants. Previously, thylakoid-bound and soluble forms of isoprene synthase had been isolated separately, each from different plant species using different procedures. Here we describe the isolation of thylakoid-bound and soluble isoprene synthases from a single willow (Salix discolor L.) leaf-fractionation protocol. Willow leaf isoprene synthase appears to be plastidic, with whole-leaf and intact chloroplast fractionations yielding approximately equal soluble (i.e. stromal) and thylakoid-bound isoprene synthase activities. Although thylakoid-bound isoprene synthase is tightly bound to the thylakoid membrane (M.C. Wildermuth, R. Fall [1996] Plant Physiol 112: 171–182), it can be solubilized by pH 10.0 treatment. The solubilized thylakoid-bound and stromal isoprene synthases exhibit similar catalytic properties, and contain essential cysteine, histidine, and arginine residues, as do other isoprenoid synthases. In addition, two regulators of foliar isoprene emission, leaf age and light, do not alter the percentage of isoprene synthase activity in the bound or soluble form. The relationship between the isoprene synthase isoforms and the implications for function and regulation of isoprene production are discussed.