Predictive Engineering of Class I Terpene Synthases Using Experimental and Computational Approaches.

Terpenoids are a highly diverse group of natural products with considerable industrial interest. Increasingly, engineered microbes are used for the production of terpenoids to replace natural extracts and chemical synthesis. Terpene synthases (TSs) show a high level of functional plasticity and are responsible for the vast structural diversity observed in natural terpenoids. Their relatively inert active sites guide intrinsically reactive linear carbocation intermediates along one of many cyclisation paths via exertion of subtle steric and electrostatic control. Due to the absence of a strong protein interaction with these intermediates, there is a remarkable lack of sequence-function relationship within the TS family, making product-outcome predictions from sequences alone challenging. This, in combination with the fact that many TSs produce multiple products from a single substrate hampers the design and use of TSs in the biomanufacturing of terpenoids. This review highlights recent advances in genome mining, computational modelling, high-throughput screening, and machine-learning that will allow more predictive engineering of these fascinating enzymes in the near future.

Molecular Determinants of Carbocation Cyclisation in Bacterial Monoterpene Synthases.

Monoterpene synthases are often promiscuous enzymes, yielding product mixtures rather than pure compounds due to the nature of the branched reaction mechanism involving reactive carbocations. Two previously identified bacterial monoterpene synthases, a linalool synthase (bLinS) and a cineole synthase (bCinS), produce nearly pure linalool and cineole from geranyl diphosphate, respectively. We used a combined experimental and computational approach to identify critical residues involved in bacterial monoterpenoid synthesis. Phe77 is essential for bCinS activity, guiding the linear carbocation intermediate towards the formation of the cyclic α-terpinyl intermediate; removal of the aromatic ring results in variants that produce acyclic products only. Computational chemistry confirmed the importance of Phe77 in carbocation stabilisation. Phe74, Phe78 and Phe179 are involved in maintaining the active site shape in bCinS without a specific role for the aromatic ring. Phe295 in bLinS, and the equivalent Ala301 in bCinS, are essential for linalool and cineole formation, respectively. Where Phe295 places steric constraints on the carbocation intermediates, Ala301 is essential for bCinS initial cyclisation and activity. Our multidisciplinary approach gives unique insights into how carefully placed amino acid residues in the active site can direct carbocations down specific paths, by placing steric constraints or offering stabilisation via cation-π interactions.

Isopentenol Utilization Pathway for the Production of Linalool in Escherichia coli Using an Improved Bacterial Linalool/Nerolidol Synthase.

Linalool is a monoterpenoid used as a fragrance ingredient, and is a promising source for alternative fuels. Synthetic biology offers attractive alternative production methods compared to extraction from natural sources and chemical synthesis. Linalool/nerolidol synthase (bLinS) from Streptomyces clavuligerus is a bifunctional enzyme, producing linalool as well as the sesquiterpenoid nerolidol when expressed in engineered Escherichia coli harbouring a precursor terpenoid pathway such as the mevalonate (MVA) pathway. Here we identified two residues important for substrate selection by bLinS, L72 and V214, where the introduction of bulkier residues results in variants with reduced nerolidol formation. Terpenoid production using canonical precursor pathways is usually limited by numerous and highly regulated enzymatic steps. Here we compared the canonical MVA pathway to the non-canonical isopentenol utilization (IU) pathway to produce linalool using the optimised bLinS variant. The IU pathway uses isoprenol and prenol to produce linalool in only five steps. Adjusting substrate, plasmid system, inducer concentration, and cell strain directs the flux towards monoterpenoids. Our integrated approach, combining enzyme engineering with flux control using the artificial IU pathway, resulted in high purity production of the commercially attractive monoterpenoid linalool, and will guide future efforts towards efficient optimisation of terpenoid production in engineered microbes.

Taming the Reactivity of Monoterpene Synthases To Guide Regioselective Product Hydroxylation.

Monoterpenoids are industrially important natural products with applications in the flavours, fragrances, fuels and pharmaceutical industries. Most monoterpenoids are produced by plants, but recently two bacterial monoterpene synthases have been identified, including a cineole synthase (bCinS). Unlike plant cineole synthases, bCinS is capable of producing nearly pure cineole from geranyl diphosphate in a complex cyclisation cascade that is tightly controlled. Here we have used a multidisciplinary approach to show that Asn305 controls water attack on the α-terpinyl cation and subsequent cyclisation and deprotonation of the α-terpineol intermediate, key steps in the cyclisation cascade which direct product formation towards cineole. Mutation of Asn305 results in variants that no longer produce α-terpineol or cineole. Molecular dynamics simulations revealed that water coordination is disrupted in all variants tested. Quantum mechanics calculations indicate that Asn305 is most likely a (transient) proton acceptor for the final deprotonation step. Our synergistic approach gives unique insight into how a single residue, Asn305, tames the promiscuous chemistry of monoterpene synthase cyclisation cascades. It does this by tightly controlling the final steps in cineole formation catalysed by bCinS to form a single hydroxylated monoterpene product.

Energy landscapes and catalysis in nitric-oxide synthase.

Nitric oxide (NO) plays diverse roles in mammalian physiology. It is involved in blood pressure regulation, neurotransmission, and immune response, and is generated through complex electron transfer reactions catalyzed by NO synthases (NOS). In neuronal NOS (nNOS), protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the heme oxygenase domain, the site of NO generation. Simple models based on crystal structures of nNOS reductase have invoked a role for large scale motions of the FMN-binding domain in shuttling electrons from the FAD-binding domain to the heme oxygenase domain. However, molecular level insight of the dynamic structural transitions in NOS enzymes during enzyme catalysis is lacking. We use pulsed electron-electron double resonance spectroscopy to derive inter-domain distance relationships in multiple conformational states of nNOS. These distance relationships are correlated with enzymatic activity through variable pressure kinetic studies of electron transfer and turnover. The binding of NADPH and calmodulin are shown to influence interdomain distance relationships as well as reaction chemistry. An important effect of calmodulin binding is to suppress adventitious electron transfer from nNOS to molecular oxygen and thereby preventing accumulation of reactive oxygen species. A complex landscape of conformations is required for nNOS catalysis beyond the simple models derived from static crystal structures of nNOS reductase. Detailed understanding of this landscape advances our understanding of nNOS catalysis/electron transfer, and could provide new opportunities for the discovery of small molecule inhibitors that bind at dynamic protein interfaces of this multidimensional energy landscape.

Decoding Catalysis by Terpene Synthases.

The review by Christianson, published in 2017 on the twentieth anniversary of the emergence of the field, summarizes the foundational discoveries and key advances in terpene synthase/cyclase (TS) biocatalysis (Christianson, D. W. Chem Rev2017, 117 (17), 11570-11648. DOI: 10.1021/acs.chemrev.7b00287). Here, we review the TS literature published since then, bringing the field up to date and looking forward to what could be the near future of TS rational design. Many revealing discoveries have been made in recent years, building on the knowledge and fundamental principles uncovered during those initial two decades of study. We use these to explore TS reaction chemistry and see how a combined experimental and computational approach helps to decipher the complexities of TS catalysis. Revealed are a suite of catalytic motifs which control product outcome in TSs, some obvious, some more subtle. We examine each in detail, using the most recent papers and insights to illustrate how exactly this fascinating class of enzymes takes a single acyclic substrate and turns it into the many thousands of complex terpenoids found in Nature. We then explore some of the recent strategies for TS engineering, including machine learning and other data-driven approaches. From this, rational and predictive engineering of TSs, "designer terpene synthases", will begin to emerge as a realistic goal.

Galactonolactone oxidoreductase from Trypanosoma cruzi employs a FAD cofactor for the synthesis of vitamin C.

Trypanosoma cruzi, the aetiological agent of Chagas' disease, is unable to salvage vitamin C (l-ascorbate) from its environment and relies on de novo synthesis for its survival. Because humans lack the capacity to synthesize ascorbate, the trypanosomal enzymes involved in ascorbate biosynthesis are interesting targets for drug therapy. The terminal step in ascorbate biosynthesis is catalyzed by flavin-dependent aldonolactone oxidoreductases belonging to the vanillyl-alcohol oxidase (VAO) protein family. Here we studied the properties of recombinant T. cruzi galactonolactone oxidoreductase (TcGAL), refolded from inclusion bodies using a reverse micelles system. The refolded enzyme shows native-like secondary structure and is active with both l-galactono-1,4-lactone and d-arabinono-1,4-lactone. At odd with an earlier claim, TcGAL employs a non-covalently bound FAD as redox-active cofactor. Moreover, it is shown for the first time that TcGAL can use molecular oxygen as electron acceptor. This is in line with the absence of a recently identified gatekeeper residue that prevents aldonolactone oxidoreductases from plants to act as oxidases.

How a 10-epi-Cubebol Synthase Avoids Premature Reaction Quenching to Form a Tricyclic Product at High Purity.

Terpenes are the largest class of natural products and are attractive targets in the fuel, fragrance, pharmaceutical, and flavor industries. Harvesting terpenes from natural sources is environmentally intensive and often gives low yields and purities, requiring further downstream processing. Engineered terpene synthases (TSs) offer a solution to these problems, but the low sequence identity and high promiscuity among TSs are major challenges for targeted engineering. Rational design of TSs requires identification of key structural and chemical motifs that steer product outcomes. Producing the sesquiterpenoid 10-epi-cubebol from farnesyl pyrophosphate (FPP) requires many steps and some of Nature's most difficult chemistry. 10-epi-Cubebol synthase from Sorangium cellulosum (ScCubS) guides a highly reactive carbocationic substrate through this pathway, preventing early quenching and ensuring correct stereochemistry at every stage. The cyclizations carried out by ScCubS potentially represent significant evolutionary expansions in the chemical space accessible by TSs. Here, we present the high-resolution crystal structure of ScCubS in complex with both a trinuclear magnesium cluster and pyrophosphate. Computational modeling, experiment, and bioinformatic analysis identified residues important in steering the reaction chemistry. We show that S206 is crucial in 10-epi-cubebol synthesis by enlisting the nearby F211 to shape the active site contour and prevent the formation of early escape cadalane products. We also show that N327 and F104 control the distribution between several early-stage cations and whether the final product is derived from the germacrane, cadalane, or cubebane hydrocarbon scaffold. Using these insights, we reengineered ScCubS so that its main product was germacradien-4-ol, which derives from the germacrane, rather than the cubebane, scaffold. Our work emphasizes that mechanistic understanding of cation stabilization in TSs can be used to guide catalytic outcomes.

Proton-coupled electron transfer in the catalytic cycle of Alcaligenes xylosoxidans copper-dependent nitrite reductase.

We demonstrated recently that two protons are involved in reduction of nitrite to nitric oxide through a proton-coupled electron transfer (ET) reaction catalyzed by the blue Cu-dependent nitrite reductase (Cu NiR) of Alcaligenes xylosoxidans (AxNiR). Here, the functionality of two putative proton channels, one involving Asn90 and the other His254, is studied using single (N90S, H254F) and double (N90S--H254F) mutants. All mutants studied are active, indicating that protons are still able to reach the active site. The H254F mutation has no effect on the catalytic activity, while the N90S mutation results in ~70% decrease in activity. Laser flash-photolysis experiments show that in H254F and wild-type enzyme electrons enter at the level of the T1Cu and then redistribute between the two Cu sites. Complete ET from T1Cu to T2Cu occurs only when nitrite binds at the T2Cu site. This indicates that substrate binding to T2Cu promotes ET from T1Cu, suggesting that the enzyme operates an ordered mechanism. In fact, in the N90S and N90S--H254F variants, where the T1Cu site redox potential is elevated by ∼60 mV, inter-Cu ET is only observed in the presence of nitrite. From these results it is evident that the Asn90 channel is the main proton channel in AxNiR, though protons can still reach the active site if this channel is disrupted. Crystallographic structures provide a clear structural rationale for these observations, including restoration of the proton delivery via a significant movement of the loop connecting the T1Cu ligands Cys130 and His139 that occurs on binding of nitrite. Notably, a role for this loop in facilitating interaction of cytochrome c(551) with Cu NiR has been suggested previously based on a crystal structure of the binary complex.

3DM: systematic analysis of heterogeneous superfamily data to discover protein functionalities.

Ten years of experience with molecular class-specific information systems (MCSIS) such as with the hand-curated G protein-coupled receptor database (GPCRDB) or the semiautomatically generated nuclear receptor database has made clear that a wide variety of questions can be answered when protein-related data from many different origins can be flexibly combined. MCSISes revolve around a multiple sequence alignment (MSA) that includes "all" available sequences from the entire superfamily, and it has been shown at many occasions that the quality of these alignments is the most crucial aspect of the MCSIS approach. We describe here a system called 3DM that can automatically build an entire MCSIS. 3DM bases the MSA on a multiple structure alignment, which implies that the availability of a large number of superfamily members with a known three-dimensional structure is a requirement for 3DM to succeed well. Thirteen MCSISes were constructed and placed on the Internet for examination. These systems have been instrumental in a large series of research projects related to enzyme activity or the understanding and engineering of specificity, protein stability engineering, DNA-diagnostics, drug design, and so forth.

Exploring novel bacterial terpene synthases.

Terpenes are the largest class of natural products with extensive structural diversity and are widely used as pharmaceuticals, herbicides, flavourings, fragrances, and biofuels. While they have mostly been isolated from plants and fungi, the availability and analysis of bacterial genome sequence data indicates that bacteria also possess many putative terpene synthase genes. In this study, we further explore this potential for terpene synthase activity in bacteria. Twenty two potential class I terpene synthase genes (TSs) were selected to represent the full sequence diversity of bacterial synthase candidates and recombinantly expressed in E. coli. Terpene synthase activity was detected for 15 of these enzymes, and included mono-, sesqui- and diterpene synthase activities. A number of confirmed sesquiterpene synthases also exhibited promiscuous monoterpene synthase activity, suggesting that bacteria are potentially a richer source of monoterpene synthase activity then previously assumed. Several terpenoid products not previously detected in bacteria were identified, including aromandendrene, acora-3,7(14)-diene and longiborneol. Overall, we have identified promiscuous terpene synthases in bacteria and demonstrated that terpene synthases with substrate promiscuity are widely distributed in nature, forming a rich resource for engineering terpene biosynthetic pathways for biotechnology.

Correlated mutation analyses on super-family alignments reveal functionally important residues.

Correlated mutation analyses (CMA) on multiple sequence alignments are widely used for the prediction of the function of amino acids. The accuracy of CMA-based predictions is mainly determined by the number of sequences, by their evolutionary distances, and by the quality of the alignments. These criteria are best met in structure-based sequence alignments of large super-families. So far, CMA-techniques have mainly been employed to study the receptor interactions. The present work shows how a novel CMA tool, called Comulator, can be used to determine networks of functionally related residues in enzymes. These analyses provide leads for protein engineering studies that are directed towards modification of enzyme specificity or activity. As proof of concept, Comulator has been applied to four enzyme super-families: the isocitrate lyase/phoshoenol-pyruvate mutase super-family, the hexokinase super-family, the RmlC-like cupin super-family, and the FAD-linked oxidases super-family. In each of those cases networks of functionally related residue positions were discovered that upon mutation influenced enzyme specificity and/or activity as predicted. We conclude that CMA is a powerful tool for redesigning enzyme activity and selectivity.

Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome.

Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavours and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclisation cascades, including the model enzyme limonene synthase (LimS). In all three mTC/S studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or α-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilisation of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the α-terpinyl cation, but is the result of an increased distance between C8 of the α-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products.

An automated pipeline for the screening of diverse monoterpene synthase libraries.

Monoterpenoids are a structurally diverse group of natural products with applications as pharmaceuticals, flavourings, fragrances, pesticides, and biofuels. Recent advances in synthetic biology offer new routes to this chemical diversity through the introduction of heterologous isoprenoid production pathways into engineered microorganisms. Due to the nature of the branched reaction mechanism, monoterpene synthases often produce multiple products when expressed in monoterpenoid production platforms. Rational engineering of terpene synthases is challenging due to a lack of correlation between protein sequence and cyclisation reaction catalysed. Directed evolution offers an attractive alternative protein engineering strategy as limited prior sequence-function knowledge is required. However, directed evolution of terpene synthases is hampered by the lack of a convenient high-throughput screening assay for the detection of multiple volatile terpene products. Here we applied an automated pipeline for the screening of diverse monoterpene synthase libraries, employing robotic liquid handling platforms coupled to GC-MS, and automated data extraction. We used the pipeline to screen pinene synthase variant libraries, with mutations in three areas of plasticity, capable of producing multiple monoterpene products. We successfully identified variants with altered product profiles and demonstrated good agreement between the results of the automated screen and traditional shake-flask cultures. In addition, useful insights into the cyclisation reaction catalysed by pinene synthase were obtained, including the identification of positions with the highest level of plasticity, and the significance of region 2 in carbocation cyclisation. The results obtained will aid the prediction and design of novel terpene synthase activities towards clean monoterpenoid products.

l-Galactono-gamma-lactone dehydrogenase from Arabidopsis thaliana, a flavoprotein involved in vitamin C biosynthesis.

l-Galactono-1,4-lactone dehydrogenase (GALDH; ferricytochrome c oxidoreductase; EC 1.3.2.3) is a mitochondrial flavoenzyme that catalyzes the final step in the biosynthesis of vitamin C (l-ascorbic acid) in plants. In the present study, we report on the biochemical properties of recombinant Arabidopsis thaliana GALDH (AtGALDH). AtGALDH oxidizes, in addition to l-galactono-1,4-lactone (K(m) = 0.17 mm, k(cat) = 134 s(-1)), l-gulono-1,4-lactone (K(m) = 13.1 mm, k(cat) = 4.0 s(-1)) using cytochrome c as an electron acceptor. Aerobic reduction of AtGALDH with the lactone substrate generates the flavin hydroquinone. The two-electron reduced enzyme reacts poorly with molecular oxygen (k(ox) = 6 x 10(2) m(-1).s(-1)). Unlike most flavoprotein dehydrogenases, AtGALDH forms a flavin N5 sulfite adduct. Anaerobic photoreduction involves the transient stabilization of the anionic flavin semiquinone. Most aldonolactone oxidoreductases contain a histidyl-FAD as a covalently bound prosthetic group. AtGALDH lacks the histidine involved in covalent FAD binding, but contains a leucine instead (Leu56). Leu56 replacements did not result in covalent flavinylation but revealed the importance of Leu56 for both FAD-binding and catalysis. The Leu56 variants showed remarkable differences in Michaelis constants for both l-galactono-1,4-lactone and l-gulono-1,4-lactone and released their FAD cofactor more easily than wild-type AtGALDH. The present study provides the first biochemical characterization of AtGALDH and some active site variants. The role of GALDH and the possible involvement of other aldonolactone oxidoreductases in the biosynthesis of vitamin C in A. thaliana are also discussed.

The growing VAO flavoprotein family.

The VAO flavoprotein family is a rapidly growing family of oxidoreductases that favor the covalent binding of the FAD cofactor. In this review we report on the catalytic properties of some newly discovered VAO family members and their mode of flavin binding. Covalent binding of the flavin is a self-catalytic post-translational modification primarily taking place in oxidases. Covalent flavinylation increases the redox potential of the cofactor and thus its oxidation power. Recent findings have revealed that some members of the VAO family anchor the flavin via a dual covalent linkage (6-S-cysteinyl-8alpha-N1-histidyl FAD). Some VAO-type aldonolactone oxidoreductases favor the non-covalent binding of the flavin cofactor. These enzymes act as dehydrogenases, using cytochrome c as electron acceptor.

Structural Basis of Catalysis in the Bacterial Monoterpene Synthases Linalool Synthase and 1,8-Cineole Synthase.

Terpenoids form the largest and stereochemically most diverse class of natural products, and there is considerable interest in producing these by biocatalysis with whole cells or purified enzymes, and by metabolic engineering. The monoterpenes are an important class of terpenes and are industrially important as flavors and fragrances. We report here structures for the recently discovered Streptomyces clavuligerus monoterpene synthases linalool synthase (bLinS) and 1,8-cineole synthase (bCinS), and we show that these are active biocatalysts for monoterpene production using biocatalysis and metabolic engineering platforms. In metabolically engineered monoterpene-producing E. coli strains, use of bLinS leads to 300-fold higher linalool production compared with the corresponding plant monoterpene synthase. With bCinS, 1,8-cineole is produced with 96% purity compared to 67% from plant species. Structures of bLinS and bCinS, and their complexes with fluorinated substrate analogues, show that these bacterial monoterpene synthases are similar to previously characterized sesquiterpene synthases. Molecular dynamics simulations suggest that these monoterpene synthases do not undergo large-scale conformational changes during the reaction cycle, making them attractive targets for structured-based protein engineering to expand the catalytic scope of these enzymes toward alternative monoterpene scaffolds. Comparison of the bLinS and bCinS structures indicates how their active sites steer reactive carbocation intermediates to the desired acyclic linalool (bLinS) or bicyclic 1,8-cineole (bCinS) products. The work reported here provides the analysis of structures for this important class of monoterpene synthase. This should now guide exploitation of the bacterial enzymes as gateway biocatalysts for the production of other monoterpenes and monoterpenoids.

Correlating Calmodulin Landscapes with Chemical Catalysis in Neuronal Nitric Oxide Synthase using Time-Resolved FRET and a 5-Deazaflavin Thermodynamic Trap.

A major challenge in enzymology is the need to correlate the dynamic properties of enzymes with, and understand the impact on, their catalytic cycles. This is especially the case with large, multicenter enzymes such as the nitric oxide synthases (NOSs), where the importance of dynamics has been inferred from a variety of structural, single-molecule, and ensemble spectroscopic approaches but where motions have not been correlated experimentally with mechanistic steps in the reaction cycle. Here we take such an approach. Using time-resolved spectroscopy employing absorbance and Förster resonance energy transfer (FRET) and exploiting the properties of a flavin analogue (5-deazaflavin mononucleotide (5-dFMN)) and isotopically labeled nicotinamide coenzymes, we correlate the timing of CaM structural changes when bound to neuronal nitric oxide synthase (nNOS) with the nNOS catalytic cycle. We show that remodeling of CaM occurs early in the electron transfer sequence (FAD reduction), not at later points in the reaction cycle (e.g., FMN reduction). Conformational changes are tightly correlated with FAD reduction kinetics and reflect a transient "opening" and then "closure" of the bound CaM molecule. We infer that displacement of the C-terminal tail on binding NADPH and subsequent FAD reduction are the likely triggers of conformational change. By combining the use of cofactor/coenzyme analogues and time-resolved FRET/absorbance spectrophotometry, we show how the reaction cycles of complex enzymes can be simplified, enabling a detailed study of the relationship between protein dynamics and reaction cycle chemistry-an approach that can also be used with other complex multicenter enzymes.

A 'Plug and Play' Platform for the Production of Diverse Monoterpene Hydrocarbon Scaffolds in Escherichia coli.

The terpenoids constitute one of the largest and most diverse classes of natural compounds with applications as pharmaceuticals, flavorings and fragrances, pesticides and biofuels. Synthetic biology is ideally placed to create new routes to this chemical diversity and facilitation of new compound discovery. The C10 monoterpenoids display a huge structural diversity produced from a single substrate, geranyl diphosphate, by a family of monoterpene cyclases and synthases (mTC/S). Here we employ a library of mTC/S in a single 'plug and play' platform system for the production of over 30 different monoterpenoids in Escherichia coli by fermentation on glucose. These products include several compounds never before produced in engineered microbes demonstrating the power of this approach to rapidly create routes to structural diversity.

Towards the free energy landscape for catalysis in mammalian nitric oxide synthases.

The general requirement for conformational sampling in biological electron transfer reactions catalysed by multi-domain redox systems has been emphasized in recent years. Crucially, we lack insight into the extent of the conformational space explored and the nature of the energy landscapes associated with these reactions. The nitric oxide synthases (NOS) produce the signalling molecule NO through a series of complex electron transfer reactions. There is accumulating evidence that protein domain dynamics and calmodulin binding are implicated in regulating electron flow from NADPH, through the FAD and FMN cofactors, to the haem oxygenase domain, where NO is generated. Simple models based on static crystal structures of the isolated reductase domain have suggested a role for large-scale motions of the FMN-binding domain in shuttling electrons from the reductase domain to the oxygenase domain. However, detailed insight into the higher-order domain architecture and dynamic structural transitions in NOS enzymes during enzyme turnover is lacking. In this review, we discuss the recent advances made towards mapping the catalytic free energy landscapes of NOS enzymes through integration of both structural techniques (e.g. cryo-electron microscopy) and biophysical techniques (e.g. pulsed-electron paramagnetic resonance). The general picture that emerges from these experiments is that NOS enzymes exist in an equilibrium of conformations, comprising a 'rugged' or 'frustrated' energy landscape, with a key regulatory role for calmodulin in driving vectorial electron transfer by altering the conformational equilibrium. A detailed understanding of these landscapes may provide new opportunities for discovery of isoform-specific inhibitors that bind at the dynamic interfaces of these multi-dimensional energy landscapes.