Search

The study of enzymes never disappoints. Despite its long history—almost 150 years following the first documented use of the word enzyme in 1878—the field of enzymology advances apace. This long journey has witnessed landmark developments that have defined modern enzymology as a broad discipline, leading to improved understanding at the molecular level, as we aspire to discover the complex relationships between enzyme structures, catalytic mechanisms and biological function. How enzymes are regulated at the gene and post‐translational levels and how catalytic activity is modulated by interactions with small ligands and macromolecules, or the broader enzyme environment, are topical areas of study. Insights from such studies guide the exploitation of natural and engineered enzymes in biomedical or industrial processes; for example, in diagnostics, pharmaceuticals manufacture and processing technologies that use immobilised enzymes and enzyme reactor‐based systems. In this Focus Issue, The FEBS Journal seeks to highlight breaking science and informative reviews, as well as personal reflections, to illustrate the breadth and importance of contemporary molecular enzymology research. [ABSTRACT FROM AUTHOR]

Biosynthetic routes based on cost‐efficient, eco‐friendly, and sustainable platforms for compounds such as styrene are urgently needed. The biosynthesis of styrene from L‐phenylalanine (L‐Phe) via trans‐cinnamate has long been established, but styrene toxicity limits yields. This study demonstrates that whole‐cell cascade biotransformation employing an E. coli consortium expressing respectively phenylalanine ammonia‐lyase and ferulic acid decarboxylase negates both the issue of styrene toxicity and the need for enzyme purification. Using resting or lyophilised cells, efficient conversion of L‐Phe to styrene (up to ∼24 g/L) is readily achieved and combined with robust extraction methods. The use of L‐Phe enriched biomass with the E. coli consortium yields an equally robust and rapid production and isolation of renewable styrene. This study establishes an improved strategy for industrial bio‐production of styrene, and by extension other toxic or reactive chemicals from corresponding bio‐compatible precursors. [ABSTRACT FROM AUTHOR]

Nitroreductases catalyse the NAD(P)H-dependent nitro reduction in nitrofuran antibiotics, which activates them into cytotoxic molecules leading to cell death. The design of new effective nitrofuran antibiotics relies on knowledge of the kinetic mechanism and nitrofuran binding mode of microbial nitroreductases NfsA and NfsB. This has been hampered by multiple co-crystallisation studies revealing ligand binding in non-electron transfer competent states. In a recent study by Day et al. (2021) the authors investigated the likely reaction mechanism and mode of nitrofurantoin binding to NfsA using potentiometry, global kinetics analysis, crystallography and molecular dynamics simulations. Their findings suggest nitrofurantoin reduction proceeds via a direct hydride transfer from reduced FMN, while the crystallographic binding orientation is an inhibitory complex. Molecular dynamics simulations suggest ligand binding orientations is dependent on the oxidation state of the FMN. This study highlights the importance of utilising computational studies alongside traditional crystallographic approaches, when multiple stable ligand binding orientations can occur. [ABSTRACT FROM AUTHOR]

Nigel Scrutton FRS is Professor of Molecular Enzymology and Biophysical Chemistry at the University of Manchester and former Director of the Manchester Institute of Biotechnology (MIB). He obtained a first degree in Biochemistry from King's College London and followed this with a PhD at the University of Cambridge. His doctoral research, undertaken in Richard Perham's laboratory, yielded fundamental breakthroughs in enzyme redesign that have stood the test of time. Nigel was awarded a ScD degree by the University of Cambridge in 2003. After faculty positions at the University of Leicester, Nigel was appointed Professor at the University of Manchester in 2005. Over the last 15 years, he has cemented his reputation as a world leader in the fields of enzyme engineering and biocatalysis, synthetic biology, biophysics and biomanufacturing, notably by establishing and directing the Synthetic Biology Research Centre 'SYNBIOCHEM' and UK Future Biomanufacturing Research Hub. In recognition of his scientific contributions, he has received many academic awards and accolades, including being elected as Fellow of the Royal Society earlier this year. In this interview, he highlights how fundamental studies of enzymatic catalysis and mechanisms are driving key advances in biotechnology and biomanufacturing, and describes how the experiences and mentors of his formative years helped to shape his successful career at the interface between discovery and application‐focused science. [ABSTRACT FROM AUTHOR]

How the dynamics of proteins assist catalysis is a contemporary issue in enzymology. In particular, this holds true for membrane‐bound enzymes, where multiple structural, spectroscopic and biochemical approaches are needed to build up a comprehensive picture of how dynamics influence enzyme reaction cycles. Of note are the recent studies of cytochrome P450 reductases (CPR)–P450 (CYP) endoplasmic reticulum redox chains, showing the relationship between dynamics and electron flow through flavin and haem redox centres and the impact this has on monooxygenation chemistry. These studies have led to deeper understanding of mechanisms of electron flow, including the timing and control of electron delivery to protein‐bound cofactors needed to facilitate CYP‐catalysed reactions. Individual and multiple component systems have been used to capture biochemical behaviour and these have led to the emergence of more integrated models of catalysis. Crucially, the effects of membrane environment and composition on reaction cycle chemistry have also been probed, including effects on coenzyme binding/release, thermodynamic control of electron transfer, conformational coupling between partner proteins and vectorial versus 'off pathway' electron flow. Here, we review these studies and discuss evidence for the emergence of dynamic structural models of electron flow along human microsomal CPR–P450 redox chains. [ABSTRACT FROM AUTHOR]

Bioengineering and synthetic biology approaches have revolutionised the field of biotechnology, enabling the introduction of non-native and de novo pathways for biofuels production. This ‘retooling’ of microorganisms is also applied to the utilisation of mixed carbon components derived from lignocellulosic biomass, a major technical barrier for the development of economically viable fermentations. This review will discuss recent advances in microorganism engineering for efficient production of alcohols from waste biomass. These advances span the introduction of new pathways to alcohols, host modifications for more cost-effective utilisation of lignocellulosic waste and modifications of existing pathways for generating new fuel additives. [ABSTRACT FROM AUTHOR]

Flavins are arguably one of the most versatile cofactors by virtue of the reactivity of the isoalloxazine ring system. A varied catalogue of reactions for the diverse family of flavoenzymes has been reported, leading to unifying concepts in (long-range) electron transfer, oxygen activation, photochemistry and substrate redox reactions. Recent examples of unprecedented flavin chemistry have been reported that uncover hidden depths of the flavoenzyme chemical repertoire. These include ring expansion of flavin through prenylation and formation of the superoxidized flavin-N5 oxide species. These and other new flavin based species are reviewed here and suggest further exciting discoveries await the flavoenzymology field. [ABSTRACT FROM AUTHOR]

Many animals are able to sense the Earth's geomagnetic field to enable behaviors such as migration. It is proposed that the magnitude and direction of the geomagnetic field modulates the activity of cryptochrome (CRY) by influencing photochemical radical pair intermediates within the protein. However, this proposal will remain theoretical until a CRY-dependent effect on a receptor neuron is shown to be modified by an external magnetic field (MF). It is established that blue-light (BL) photoactivation of CRY is sufficient to depolarize and activate Drosophila neurons. Here, we show that this CRY-dependent effect is significantly potentiated in the presence of an applied MF (100 mT). We use electrophysiological recordings from larval identified motoneurons, in which CRY is ectopically expressed, to show that BL-dependent depolarization of membrane potential and increased input resistance are markedly potentiated by an MF. Analysis of membrane excitability shows that these effects of MF exposure evoke increased action potential firing. Almost nothing is known about the mechanism by which a magnetically induced change in CRY activity might produce a behavioral response. We further report that specific structural changes to the protein alter the impact of the MF in ways that are strikingly similar to those from recent behavioral studies into the magnetic sense of Drosophila. These observations provide the first direct experimental evidence to support the hypothesis that MF modulation of CRY activity is capable of influencing neuron activity to allow animal magnetoreception. [ABSTRACT FROM AUTHOR]

Throughout nature, both free radicals and transient radical reaction intermediates are vital to many biological functions. Coenzyme B12 is a case in point. This organometallic cofactor generates a radical pair upon activation in its dependent enzymes by substrate binding and following photolysis. The resulting cob(ii)alamin/5′-deoxyadenosyl radical pair has unusual magnetic properties that present a challenge to detailed investigation at ambient temperatures. Here, we use femtosecond transient absorption spectroscopy adapted for magnetic field exposure to reveal that the spin dynamics of the B12 radical pair are sufficiently fast for magnetic field effects to be observed on the ultrafast reaction kinetics. Moreover, the large difference in g-values between the radicals of the pair means that effects of the Δg mechanism are observed for the first time for a radical pair system exposed to magnetic fields below 1 T. Spin dynamic simulations allow a value of the cob(ii)alamin radical g-value (2.105) at ambient temperature to be extracted and, because the spin dynamic time scale is faster than the diffusional rotation of the cob(ii)alamin radical, the observed value corresponds to the anisotropic g|| value for this radical. [ABSTRACT FROM AUTHOR]

A computational study was performed on the experimentally elusive cyclisation step in the cofactor pyridoxal 5′-phosphate (PLP)-dependent D-ornithine 4,5-aminomutase (OAM)-catalysed reaction. Calculations using both model systems and a combined quantum mechanics/molecular mechanics approach suggest that regulation of the cyclic radical intermediate is achieved through the synergy of the intrinsic catalytic power of cofactor PLP and the active site of the enzyme. The captodative effect of PLP is balanced by an enzyme active site that controls the deprotonation of both the pyridine nitrogen atom (N1) and the Schiff-base nitrogen atom (N2). Furthermore, electrostatic interactions between the terminal carboxylate and amino groups of the substrate and Arg297 and Glu81 impose substantial 'strain' energy on the orientation of the cyclic intermediate to control its trajectory. In addition the 'strain' energy, which appears to be sensitive to both the number of carbon atoms in the substrate/analogue and the position of the radical intermediates, may play a key role in controlling the transition of the enzyme from the closed to the open state. Our results provide new insights into several aspects of the radical mechanism in aminomutase catalysis and broaden our understanding of cofactor PLP-dependent reactions. [ABSTRACT FROM AUTHOR]

Highlights: [•] Old Yellow Enzymes are primarily used for biocatalytic hydrogenations. [•] Medium chain dehydrogenase/reductases can substitute for OYEs. [•] Unsaturated ester reduction can occur with extra electron withdrawing substituents. [•] Chemoenzymatic or biocatalytic multienzyme cascading reactions are possible. [•] Optimisation of biocatalytic processes can improve commercial viability. [Copyright &y& Elsevier]

Fast motions (femtosecond to picosecond) and their potential involvement during enzyme-catalysed reactions have ignited considerable interest in recent years. Their influence on reaction chemistry has been inferred indirectly from studies of the anomalous temperature dependence of kinetic isotope effects and computational simulations. But can such motion reduce the width and height of energy barriers along the reaction coordinate, and contribute to quantum mechanical and/or classical nuclear-transfer chemistry? Here we discuss contemporary ideas for enzymatic reactions invoking a role for fast 'promoting' (or 'compressive') motions that, in principle, can aid hydrogen-transfer reactions. Of key importance is the direct demonstration of a role for compressive motions and the ability to understand in atomic detail the structural origin of these fast motions, but so far this has not been achieved. Here we discuss both indirect experimental evidence that supports a role for compressive motion and the additional insight gained from computational simulations. [ABSTRACT FROM AUTHOR]

Many enzymes use one or more cofactors, such as biotin, heme, or flavin. These cofactors may be bound to the enzyme in a noncovalent or covalent manner. Although most flavoproteins contain a noncovalently bound flavin cofactor (FMN or FAD), a large number have these cofactors covalently linked to the polypeptide chain. Most covalent flavin–protein linkages involve a single cofactor attachment via a histidyl, tyrosyl, cysteinyl or threonyl linkage. However, some flavoproteins contain a flavin that is tethered to two amino acids. In the last decade, many studies have focused on elucidating the mechanism(s) of covalent flavin incorporation (flavinylation) and the possible role(s) of covalent protein–flavin bonds. These endeavors have revealed that covalent flavinylation is a post-translational and self-catalytic process. This review presents an overview of the known types of covalent flavin bonds and the proposed mechanisms and roles of covalent flavinylation. [ABSTRACT FROM AUTHOR]

Human methionine synthase reductase (MSR), a diflavin enzyme, restores the activity of human methionine synthase through reductive methylation of methionine synthase (MS)-bound cob(II)alamin. Recently, it was also reported that MSR enhances uptake of cobalamin by apo-MS, a role associated with the MSR-catalysed reduction of exogenous aquacob(III)alamin to cob(II)alamin [Yamada K, Gravel RA, TorayaT & Matthews RG (2006) Proc Natl Acad Sci USA 103, 9476–9481]. Here, we report the expression and purification of human methionine synthase from Pichia pastoris. This has enabled us to assess the ability of human MSR and two other structurally related diflavin reductase enzymes (cytochrome P450 reductase and the reductase domain of neuronal nitric oxide synthase) to: (a) stimulate formation of holo-MS from aquacob(III)alamin and the apo-form of MS; and (b) reactivate the inert cob(II)alamin form of MS that accumulates during enzyme catalysis. Of the three diflavin reductases studied, cytochrome P450 reductase had the highest turnover rate (55.5 s−1) for aquacob(III)alamin reduction, and the reductase domain of neuronal nitric oxide synthase elicited the highest specificity ( kcat/ Km of 1.5 × 105 m−1·s−1) and MSR had the lowest Km (6.6 μm) for the cofactor. Despite the ability of all three enzymes to reduce aquacob(III)alamin, only MSR (the full-length form or the isolated FMN domain) enhanced the uptake of cobalamin by apo-MS. MSR was also the only diflavin reductase to reactivate the inert cob(II)alamin form of purified human MS ( Kact of 107 nm) isolated from Pichia pastoris. Our work shows that reactivation of cob(II)alamin MS and incorporation of cobalamin into apo-MS is enhanced through specific protein–protein interactions between the MSR FMN domain and MS. [ABSTRACT FROM AUTHOR]

ABSTRACT: Hydrostatic pressure offers an alternative to temperature as an experimental probe of hydrogentransfer reactions. H tunneling reactions have been shown to exhibit kinetic isotope effects (KIEs) that are sensitive to pressure, and environmentally coupled H tunneling reactions, those reactions in which H transfer is coupled to atomic fluctuations (a promoting vibration) along the reaction coordinate, often have quite temperature-dependent KIEs. We present here a theoretical treatment of the combined effect of temperature and pressure on environmentally coupled H tunneling reactions. We develop a generalized expression for the KIE, which can be used as a simple fitting function for combined experimental temperatureand pressure-dependent KIE data sets. With this expression, we are able to extract information about the pressure dependence of both the apparent tunneling distance and the frequency of the promoting vibration. The KIE expression is tested on two data sets ( the reduction of chioranil by leuco crystal violet [Isaacs, N. S., Javaid, K., and Rannala, E. (1998) J. Chem. Soc., Perkin Trans. 2, 709-711] and the reduction of morphinone reductase by NADH [Hay, S., Sutcliffe, M. J., and Scrutton, N. S. (2007) Proc. Nati. Acad. Sci. U.S.A. 104, 507-512] ) and suggests that hydrostatic pressure is a sensitive probe of nuclear quantum mechanical effects in H-transfer reactions. [ABSTRACT FROM AUTHOR]

Methionine synthase (MS) is a cobalamin-dependent enzyme. It transfers a methyl group from methyltetrahydrofolate to homocysteine forming methionine and tetrahydrofolate. On the basis of sequence similarity with Escherichia coli cobalamin-dependent MS (MetH), human MS comprises four discrete functional modules that bind from the N- to C-terminus, respectively, homocysteine, methyltetrahydrofolate, cobalamin, and S-adenosylmethionine (AdoMet). The C-terminal activation domain also interacts with methionine synthase reductase (MSR), a NADPH-dependent diflavin oxidoreductase required for the reductive regeneration of catalytically inert cob(II)alamin (which is formed every 200-1000 catalytic cycles of MS) to cob(I)alamin. We have investigated complex formation between the (i) MS activation domain and MSR and (ii) MS activation domain and the isolated FMN-binding domain of MSR. We show that the MS activation domain interacts directly with the FMN-binding domain of MSR. Binding is weakened at high ionic strength, emphasizing the importance of electrostatic interactions at the protein—protein interface. Mutagenesis of conserved lysine residues (Lys1071 and Lys987) in the human activation domain weakens this protein interaction. Chemical cross-linking demonstrates complex formation mediated by acidic residues (FMN-binding domain) and basic residues (activation domain). The activation domain and isolated FMN-domain form a 1:1 complex, but a 1:2 complex is formed with activation domain and MSR. The midpoint reduction potentials of the FAD and FMN cofactors of MSR are not perturbed significantly on forming this complex, implying that electron transfer to cob(II)alamin is endergonic. The kinetics of electron transfer in MSR and the MSR—activation domain complex are similar. Our studies indicate (i) conserved binding determinants, but differences in protein stoichiometry, between human MS and bacterial MetH in complex formation with redox partners; (ii) a substantial endergonic barrier to electron transfer in the reactivation complex; and (iii) a lack of control on the thermodynamics and kinetics of electron transfer in MSR exerted by complex formation with activation domain. The structural and functional consequences of complex formation are discussed in light of the known crystal structure of human activation domain and the inferred conformational heterogeneity of the multidomain MSR—MS complex. [ABSTRACT FROM AUTHOR]

The quinoprotein aromatic amine dehydrogenase (AADH) uses a covalently bound tryptophan tryptophyiquinone (TTQ) cofactor to oxidatively deaminate primary aromatic amines. Recent crystal structures have provided insight into the reductive half-reaction. In contrast, no atomic details are available for the oxidative half-reaction. The TTQ O7 hydroxyl group is protonated during reduction, but it is unclear how this proton can be removed during the oxidative half-reaction. Furthermore, compared with the electron transfer from the N-quinol form, electron transfer from the non-physiological O-quinol form to azurin is significantly slower. Here we report crystal structures of the O-quinol, N-quinol, and N-semiquinone forms of AADH. A comparison of oxidized and substrate reduced AADH species reveals changes in the TTQ-containing subunit, extending from residues in the immediate vicinity of the N-quinol to the putative azurin docking site, suggesting a mechanism whereby TTQ redox state influences interprotein electron transfer. In contrast, chemical reduction of the TTQ center has no significant effect on protein conformation. Furthermore, structural reorganization upon substrate reduction places a water molecule near TTQ 07 where it can act as proton acceptor. The structure of the N-semiquinone, however, is essentially similar to oxidized AADH. Surprisingly, in the presence of substrate a covalent N-semiquinone substrate adduct is observed. To our knowledge this is the first detailed insight into a complex, branching mechanism of quinone oxidation where significant structural reorganization upon reduction of the quinone center directly influences formation of the electron transfer complex and nature of the electron transfer process. [ABSTRACT FROM AUTHOR]

The rate of electron transfer by tunnelling decreases exponentially with distance, but is generally not rate limiting for distances up to 14Å, enabling the robust design of redox systems. Fast transfer over distances greater than 14Å is accomplished using diffusible electron carriers, an array of closely spaced redox centres or large-scale motion of a redox-active domain. Recent advances indicate that all three mechanisms are used in interprotein electron transfer. The classic stratagem, diffusible electron carriers, may be extended with either of the other designs. The use of an array of solvent-exposed, closely spaced redox centres can maximise productive collisions. Also, the use of conformational sampling via domain motion within the electron transfer complex optimises tunnelling probability. [Copyright &y& Elsevier]

Human methionine synthase reductase (MSR) is a key enzyme in folate and methionine metabolism as it reactivates the catalytically inert cob(II)alamin form of methionine synthase (MS). Electron transfer from MSR to the cob(II)alamin cofactor coupled with methyl transfer from S-adenosyl methionine returns MS to the active methylcob(III)alamin state. MSR contains stoichiometric amounts of FAD and FMN, which shuttle NADPH-derived electrons to the MS cob(II)alamin cofactor. Herein, we have investigated the pre-steady state kinetic behavior of the reductive half-reaction of MSR by anaerobic stopped-flow absorbance and fluorescence spectroscopy. Photodiode array and single-wavelength spectroscopy performed on both full-length MSR and the isolated FAD domain enabled assignment of observed kinetic phases to mechanistic steps in reduction of the flavins. Under single turnover conditions, reduction of the isolated FAD domain by NADPH occurs in two kinetically resolved steps: a rapid (120 s[sup-1]) phase, characterized by the formation of a charge-transfer complex between oxidized FAD and NADPH, is followed by a slower (20 s[sup-1]) phase involving flavin reduction. These two kinetic phases are also observed for reduction of full-length MSR by NADPH, and are followed by two slower and additional kinetic phases (0.2 and 0.016 s[sup-1]) involving electron transfer between FAD and FMN (thus yielding the disemiquinoid form of MSR) and further reduction of MSR by a second molecule of NADPH. The observed rate constants associated with flavin reduction are dependent hyperbolically on NADPH and [4(R)-[sup2]H]- NADPH concentration, and the observed primary kinetic isotope effect on this step is 2.2 and 1.7 for the isolated FAD domain and full-length MSR, respectively. Both full-length MSR and the separated FAD domain that have been reduced with dithionite catalyze the reduction of NADP[sup+]. [ABSTRACT FROM AUTHOR]

Cys-999 is one component of a triad (Cys-999, Ser-830, and Asp-1044) located in the FAD domain of flavocytochrome P450 BM3 that is almost entirely conserved throughout the diflavin reductase family of enzymes. The role of Cys-999 has been studied by steady-state kinetics, stopped-flow spectroscopy, and potentiometry. The C999A mutants of BM3 reductase (containing both FAD and FMN cofactors) and the isolated FAD domain are substantially compromised in their capacity to reduce artificial electron acceptors in steady-state turnover with either NADPH or NADH aS electron donors. Stoppedflow studies indicate that this is due primarily to a substantially slower rate of hydride transfer from nicotinamide coenzyme to FAD cofactor in the C999A enzymes. The compromised rates of hydride transfer are not attributable to altered thermodynamic properties of the flavins. A reduced enzyme-NADP[sup +] chargetransfer species is populated following hydride transfer in the wild-type FAD domain, consistent with the slow release of NADP[sup +] from the 2-electron-reduced enzyme. This intermediate does not accumulate in the C999A FAD domain or wild-type and C999A BM3 reductases, suggesting more rapid release of NADP[sup +] from these enzyme forms. Rapid internal electron transfer from FAD to FMN in wild-type BM3 reductase releases NADP[sup +] from the nicotinamide-binding site, thus preventing the inhibition of enzyme activity through the accumulation of a stable FADH[sub 2]-NADP[sup +] charge-transfer complex. Hydride transfer is reversible, and the observed rate of oxidation of the 2-electron-reduced C999A BM3 reductase and FAD domain is hyperbolically dependent on NADP[sup +] concentration. With the wild-type BM3 reductase and FAD domain, the rate of flavin oxidation displays an unusual dependence on NADP[sup +] concentration, consistent with a two-site binding model in which two coenzyme molecules bind to catalytic and regulatory regions (or sites) within a bipartite coenzyme binding site. A kinetic model is proposed in which binding of coenzyme to the regulatory site hinders sterically the release of NADPH from the catalytic site. The results are discussed in the light of kinetic and structural studies on mammalian cytochrome P450 reductase. [ABSTRACT FROM AUTHOR]

Recent years have witnessed high levels of activity in identifying enzyme systems that catalyse H-transfer by quantum tunneling. Rather than being restricted to a small number of specific enzymes as perceived initially, it has now become an accepted mechanism for H-transfer in a growing number of enzymes. Furthermore, H-tunneling is driven by the thermally induced dynamics of the enzyme. In some of those enzymes that break stable C–H bonds the reaction proceeds purely by quantum tunneling, without the need to partially ascend the barrier. Enzymes studied that fall into this category include the flavoprotein and quinoprotein amine dehydrogenases, which have proved to be excellent model systems. These enzymes have enabled us to study the relationship between barrier shape and reaction kinetics. This has involved studies with ‘slow’ and ‘fast’ substrates and enzymes impaired by mutagenesis. A number of key questions now remain, including the nature of the coupling between protein dynamics and quantum tunneling. The wide-ranging implications of quantum tunneling introduce a paradigm shift in the conceptual framework for enzyme catalysis, inhibition and design. [ABSTRACT FROM AUTHOR]

Provides information on a study which examine how enzymes achieve high catalytic rates by evolving complementarity with the transition state of the substrate. Description of the over-the-barrier catalysis; Discussion of tunneling through-the-barrier; Description of the quantum tunneling facilitated by protein dynamics.

A British molecular biologist who made groundbreaking contributions to the molecular structure, assembly and function of proteins and was Editor ‐ in ‐ Chief of The FEBS Journal (1998–2013). [ABSTRACT FROM AUTHOR]

The circular economy is anticipated to bring a disruptive transformation in manufacturing technologies. Robust and industrial scalable microbial strains that can simultaneously assimilate and valorize multiple carbon substrates are highly desirable, as waste bioresources contain substantial amounts of renewable and fermentable carbon, which is diverse. Lignocellulosic biomass (LCB) is identified as an inexhaustible and alternative resource to reduce global dependence on oil. Glucose, xylose, and arabinose are the major monomeric sugars in LCB. However, primary research has focused on the use of glucose. On the other hand, the valorization of pentose sugars, xylose, and arabinose, has been mainly overlooked, despite possible assimilation by vast microbial communities. The present review highlights the research efforts that have explicitly proven the suitability of arabinose as the starting feedstock for producing various chemical building blocks via biological routes. It begins by analyzing the availability of various arabinose-rich biorenewable sources that can serve as potential feedstocks for biorefineries. The subsequent section outlines the current understanding of arabinose metabolism, biochemical routes prevalent in prokaryotic and eukaryotic systems, and possible products that can be derived from this sugar. Further, currently, exemplar products from arabinose, including arabitol, 2,3-butanediol, 1,2,3-butanetriol, ethanol, lactic acid, and xylitol are discussed, which have been produced by native and non-native microbial strains using metabolic engineering and genome editing tools. The final section deals with the challenges and obstacles associated with arabinose-based production, followed by concluding remarks and prospects. [ABSTRACT FROM AUTHOR]

Microbial instability is a common problem during bio-production based on microbial hosts. Halomonas bluephagenesis has been developed as a chassis for next generation industrial biotechnology (NGIB) under open and unsterile conditions. However, the hidden genomic information and peculiar metabolism have significantly hampered its deep exploitation for cell-factory engineering. Based on the freshly completed genome sequence of H. bluephagenesis TD01, which reveals 1889 biological process-associated genes grouped into 84 GO-slim terms. An enzyme constrained genome-scale metabolic model Halo-ecGEM was constructed, which showed strong ability to simulate fed-batch fermentations. A visible salt-stress responsive landscape was achieved by combining GO-slim term enrichment and CVT-based omics profiling, demonstrating that cells deploy most of the protein resources by force to support the essential activity of translation and protein metabolism when exposed to salt stress. Under the guidance of Halo-ecGEM, eight transposases were deleted, leading to a significantly enhanced stability for its growth and bioproduction of various polyhydroxyalkanoates (PHA) including 3-hydroxybutyrate (3HB) homopolymer PHB, 3HB and 3-hydroxyvalerate (3HV) copolymer PHBV, as well as 3HB and 4-hydroxyvalerate (4HB) copolymer P34HB. This study sheds new light on the metabolic characteristics and stress-response landscape of H. bluephagenesis , achieving for the first time to construct a long-term growth stable chassis for industrial applications. For the first time, it was demonstrated that genome encoded transposons are the reason for microbial instability during growth in flasks and fermentors. [Display omitted] • 84 GO-slim terms were classified to study the stress response of Halomonas. • A translation-dependent stress regulation pattern of Halomonas was uncovered. • Halo-ecGEM was developed for fermentation simulation with good fit. • A growth stable chassis WZY278ΔTns was developed aftter 8 transposases deletion. [ABSTRACT FROM AUTHOR]

Examines the ground state tunnelling driven by protein dynamics. Catalysis of enzyme; Role of enzymatic Hydrogen-transfer to wave-particle duality of matter; Activation of energies.

The interaction between two physiological redox partners, trimethylamine dehydrogenase and electron-transferring flavoprotein, has been characterized quantitatively by analytical ultracentrifugation at 4°C. Analysis of sedimentation-equilibrium distributions obtained at 15 000 rpm for mixtures in 10 mM potassium phosphate, pH 7.5, by means of the psi function [Wills, P. R., Jacobsen, M. E & Winzor, D. J. (1996) Biopolymers 38, 119-1301 has yielded an intrinsic dissociation constant of 3-7 µM for the interaction of electron-transferring flavoprotein with two equivalent and independent sites on the homodimeric enzyme. This investigation indicates the potential of sedimentation equilibrium for the quantitative characterization of interactions between dissimilar macromolecules. [ABSTRACT FROM AUTHOR]

States that when glycine of Escherichia coli glutahione is replaced by a bulky tryptophan residue, the enzyme becomes highly cooperative for glutathione binding; Two principal models advanced to explain cooperative bahavior; Cooperativity for glutathione binding and loss of cooperativity when mutant subunit is hybridized with a wild-type subunit to create a heterodimer.

Covering: up to 2021 Terpenoids are a diverse group of chemicals used in a wide range of industries. Microbial terpenoid production has the potential to displace traditional manufacturing of these compounds with renewable processes, but further titre improvements are needed to reach cost competitiveness. This review discusses strategies to increase terpenoid titres in Escherichia coli with a focus on alternative metabolic pathways. Alternative pathways can lead to improved titres by providing higher orthogonality to native metabolism that redirects carbon flux, by avoiding toxic intermediates, by bypassing highly-regulated or bottleneck steps, or by being shorter and thus more efficient and easier to manipulate. The canonical 2-C-methyl- D -erythritol 4-phosphate (MEP) and mevalonate (MVA) pathways are engineered to increase titres, sometimes using homologs from different species to address bottlenecks. Further, alternative terpenoid pathways, including additional entry points into the MEP and MVA pathways, archaeal MVA pathways, and new artificial pathways provide new tools to increase titres. Prenyl diphosphate synthases elongate terpenoid chains, and alternative homologs create orthogonal pathways and increase product diversity. Alternative sources of terpenoid synthases and modifying enzymes can also be better suited for E. coli expression. Mining the growing number of bacterial genomes for new bacterial terpenoid synthases and modifying enzymes identifies enzymes that outperform eukaryotic ones and expand microbial terpenoid production diversity. Terpenoid removal from cells is also crucial in production, and so terpenoid recovery and approaches to handle end-product toxicity increase titres. Combined, these strategies are contributing to current efforts to increase microbial terpenoid production towards commercial feasibility. [ABSTRACT FROM AUTHOR]

The photoenzyme protochlorophyllide oxidoreductase (POR) is an important enzyme for understanding biological H‐transfer mechanisms. It uses light to catalyse the reduction of protochlorophyllide to chlorophyllide, a key step in chlorophyll biosynthesis. Although a wealth of spectroscopic data have provided crucial mechanistic insight, a structural rationale for POR photocatalysis has proved challenging and remains hotly debated. Recent structural models of the ternary enzyme–substrate complex, derived from crystal and electron microscopy data, show differences in the orientation of the protochlorophyllide substrate and the architecture of the POR active site, with significant implications for the catalytic mechanism. Here, we use a combination of computational and experimental approaches to investigate the compatibility of each structural model with the hypothesised reaction mechanisms and propose an alternative structural model for the cyanobacterial POR ternary complex. We show that a strictly conserved tyrosine, previously proposed to act as the proton donor in POR photocatalysis, is unlikely to be involved in this step of the reaction but is crucial for Pchlide binding. Instead, an active site cysteine is important for both hydride and proton transfer reactions in POR and is proposed to act as the proton donor, either directly or through a water‐mediated network. Moreover, a conserved glutamine is important for Pchlide binding and ensuring efficient photochemistry by tuning its electronic properties, likely by interacting with the central Mg atom of the substrate. This optimal 'binding pose' for the POR ternary enzyme–substrate complex illustrates how light energy can be harnessed to facilitate enzyme catalysis by this unique enzyme. [ABSTRACT FROM AUTHOR]

The article discusses the study that describes an algal photoenzyme using blue light to convert fatty acids into hydrocarbons. It provides overview of the process of the study and the role of photoenzyme which uses a riboflavin cofactor to drive fatty acid decarboxylation. It outlines the result which suggests that the optimization of latent photochemistry in flavoprotein biocatalysis will become an area of intense research activity.

• The majority of tryptophan is metabolised through the kynurenine pathway in mammals. • Several kynurenine metabolites are neuroactive and modulate inflammation. • The kynurenine pathway is activated in patients with COVID-19. • Compounds have been developed that modulate kynurenine pathway by inhibiting kynurenine3-monooxygenase. • These inhibitors may be beneficial in COVID-19 by reducing inflammation. The novel respiratory virus severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), which causes coronavirus disease 2019 (COVID-19), emerged during late 2019 and spread rapidly across the world. It is now recognised that the nervous system can be affected in COVID-19, with several studies reporting long-term cognitive problems in patients. The metabolic pathway of tryptophan degradation, known as the kynurenine pathway (KP), is significantly activated in patients with COVID-19. KP metabolites have roles in regulating both inflammatory/immune responses and neurological functions. In this review, we speculate on the effects of KP activation in patients with COVID-19, and how modulation of this pathway might impact inflammation and reduce neurological symptoms. The kynurenine pathway of tryptophan degradation is activated in patients with Coronavirus 2019 (COVID-19). The possible effects of kynurenine metabolites on inflammation and neurocognition in COVID-19 and the potential use of kynurenine 3-monooxygenase inhibitors for treatment are discussed. [ABSTRACT FROM AUTHOR]

Ectoine, a crucial osmoprotectant for salt adaptation in halophiles, has gained growing interest in cosmetics and medical industries. However, its production remains challenged by stringent fermentation process in model microorganisms and low production level in its native producers. Here, we systematically engineered the native ectoine producer Halomonas bluephagenesis for ectoine production by overexpressing ectABC operon, increasing precursors availability, enhancing product transport system and optimizing its growth medium. The final engineered H. bluephagenesis produced 85 g/L ectoine in 52 h under open unsterile incubation in a 7 L bioreactor in the absence of plasmid, antibiotic or inducer. Furthermore, it was successfully demonstrated the feasibility of decoupling salt concentration with ectoine synthesis and co-production with bioplastic P(3HB- co -4HB) by the engineered H. bluephagenesis. The unsterile fermentation process and significantly increased ectoine titer indicate that H. bluephagenesis as the chassis of Next-Generation Industrial Biotechnology (NGIB), is promising for the biomanufacturing of not only intracellular bioplastic PHA but also small molecular compound such as ectoine. • Ectoine hyperproduction was achieved by systematically engineering a native producer Halomonas bluephagenesis. • 85 g/L ectoine was produced in a 7 L non-sterile fed-batch fermentation without plasmid, antibiotic, or inducer. • Dependence of ectoine synthesis and NaCl concentration was removed in the H. bluephagenesis. • Bioplastic P(3HB- co -4HB) and ectoine was co-produced by engineered H. bluephagenesis. [ABSTRACT FROM AUTHOR]

The development of sustainable routes to the bio-manufacture of gaseous hydrocarbons will contribute widely to future energy needs. Their realisation would contribute towards minimising over-reliance on fossil fuels, improving air quality, reducing carbon footprints and enhancing overall energy security. Alkane gases (propane, butane and isobutane) are efficient and clean-burning fuels. They are established globally within the transportation industry and are used for domestic heating and cooking, non-greenhouse gas refrigerants and as aerosol propellants. As no natural biosynthetic routes to short chain alkanes have been discovered, de novo pathways have been engineered. These pathways incorporate one of two enzymes, either aldehyde deformylating oxygenase or fatty acid photodecarboxylase, to catalyse the final step that leads to gas formation. These new pathways are derived from established routes of fatty acid biosynthesis, reverse β-oxidation for butanol production, valine biosynthesis and amino acid degradation. Single-step production of alkane gases in vivo is also possible, where one recombinant biocatalyst can catalyse gas formation from exogenously supplied short-chain fatty acid precursors. This review explores current progress in bio-alkane gas production, and highlights the potential for implementation of scalable and sustainable commercial bioproduction hubs. [ABSTRACT FROM AUTHOR]

This FEBS Journal Special Issue showcases the latest findings in the field of Flavins and Flavoproteins. Two reviews summarize ultrafast fluorescence techniques for studying flavoenezymes and protein dynamics of nitric oxide synthase. Research articles include studies of oxidases, oxygenase and dehydrogenase, electron transfer flavoprotein, photoreceptors, enzyme inhibition, flavoenzyme for bioconversion, regulation of riboflavin biosynthesis, and the use of physical principles for interpreting protein dynamics. [ABSTRACT FROM AUTHOR]

Background: Production of relatively low value, bulk commodity chemicals and fuels by microbial species requires a step-change in approach to decrease the capital and operational costs associated with scaled fermentation. The utilisation of the robust and halophilic industrial host organisms of the genus Halomonas could dramatically decrease biomanufacturing costs owing to their ability to grow in seawater, using waste biogenic feedstocks, under non-sterile conditions. Results: We describe the isolation of Halomonas rowanensis, a novel facultative chemoautotrophic species of Halomonas from a natural brine spring. We investigated the ability of this species to produce ectoine, a compound of considerable industrial interest, under heterotrophic conditions. Fixation of radiolabelled NaH14CO3 by H. rowanensis was confirmed in mineral medium supplied with thiosulfate as an energy source. Genome sequencing suggested carbon fixation proceeds via a reductive tricarboxylic acid cycle, and not the Calvin–Bensen–Bassham cycle. The mechanism of energy generation to support chemoautotrophy is unknown owing to the absence of an annotated SOX-based thiosulfate-mediated energy conversion system. We investigated further the biotechnological potential of the isolated H. rowanensis by demonstrating production of the gaseous hydrocarbon (bio-propane), bioplastics (poly-3-hydroxybutyrate) and osmolytes (ectoine) under heterotrophic and autotrophic CO2 fixation growth conditions. Conclusions: This proof-of-concept study illustrates the value of recruiting environmental isolates as industrial hosts for chemicals biomanufacturing, where CO2 utilisation could replace, or augment, the use of biogenic feedstocks in non-sterile, industrialised bioreactors. Highlights: New halophilic isolate Halomonas rowanensis is useful for chemicals and fuel production at neutral pH. H. rowanensis is a likely facultative chemolithotroph capable of CO2 fixation in the presence of thiosulfate. Propane production occurred under both heterotrophic and autotrophic growth conditions with recombinant H. rowanensis. Potential application of H. rowanensis in the non-sterile production of chemicals and advanced synthetic fuels. [ABSTRACT FROM AUTHOR]

[Display omitted] • Review focusing on the applications of enzymes in the synthesis of renewable monomers. • Covering cascade reactions including chemo-enzymatic processes to obtain bio-based drop-in chemicals or novel compounds for used in biodegradable polymers. • Perspective on current challenges, commercial viability and outlook of industrial biotechnology in the polymer/material industry. Advancements in directed evolution and genome sequencing have led to the discovery and evolution of enzymes with new and improved catalytic properties. The pharmaceutical industry has greatly benefited from this, evidenced by the number of publications and manufacturing processes reported in the last decade. However, biocatalysts are rarely reported in the polymer industry which has prompted the writing of this review to highlight enzymatic processes towards the production of structurally diverse monomers. These highlights detail the process development, evolution and novel use of enzymes in polymer synthesis for medical, packaging and coating applications. The review features the use of four enzyme classes, oxidoreductases, transferases, hydrolases and lyases in the synthesis of high-value monomers found in commercial and next-generation biodegradable polymers. [ABSTRACT FROM AUTHOR]

The article presents information on a study which determines the magnetic field effects in the haem-dependent enzyme horseradish peroxidase (HRP). The study shows that the application of external magnetic fields can perturb reaction kinetics through the constraints of the radical pair mechanism. The study findings suggest that relatively modest fields, including those of environmentally realistic magnitude, can influence the yields and rates of radical pair. According to the researchers, the reaction catalyzed by HRP includes oxidative and reductive half reactions.

Research on enzyme mechanisms is advancing knowledge of the chemistry and biochemistry of catalytic mechanisms by biological systems. The structural‐dynamical properties of enzymes are of key importance. Advanced methodological approaches and new insights into enzyme functioning, and new emerging approaches for the redesign/design of enzymes or artificial biocatalysts, were discussed at the EMBO conference on Catalytic Mechanisms by Biological Systems. [ABSTRACT FROM AUTHOR]

This perspective reviews single molecule and ensemble fluorescence spectroscopy studies of the three tissue specific nitric oxide synthase (NOS) isoenzymes and the related diflavin oxidoreductase cytochrome P450 reductase. The focus is on the role of protein dynamics and the protein conformational landscape and we discuss how recent fluorescence-based studies have helped in illustrating how the nature of the NOS conformational landscape relates to enzyme turnover and catalysis. [ABSTRACT FROM AUTHOR]

Halomonas bluephagenesis TD1.0 was engineered to produce the biofuel propane, bioplastic poly‐3‐hydroxybutyrate (PHB), and biochemicals mandelate and hydroxymandelate in a single, semi‐continuous batch fermentation under non‐sterile conditions. Multi‐product separation was achieved by segregation of the headspace gas (propane), fermentation broth ([hydroxy]mandelate) and cellular biomass (PHB). Engineering was performed by incorporating the genes encoding fatty acid photodecarboxylase (CvFAP) and hydroxymandelic acid synthase (SyHMAS) into a H. bluephagenesis hmgCAB cassette knockout to channel flux towards (hydroxy)mandelate. Design of Experiment strategies were coupled with fermentation trials to simultaneously optimize each product. Propane and mandelate titres were the highest reported for H. bluephagenesis (62 g/gDCW and 71 ± 10 mg/L respectively) with PHB titres (69% g/gDCW) comparable to other published studies. This proof‐of‐concept achievement of four easily separated products within one fermentation is a novel achievement probing the versatility of biotechnology, further elevating H. bluephagenesis as a Next Generation Industrial Biotechnology (NGIB) chassis by producing highly valued products at a reduced cost. [ABSTRACT FROM AUTHOR]

Cannabinoids are a therapeutically valuable class of secondary metabolites with a vast number of substituents. The native cannabinoid biosynthetic pathway of Cannabis sativa generates cannabigerolic acid (CBGA), the common substrate to multiple cannabinoid synthases. The bioactive decarboxylated analog of this compound, cannabigerol (CBG), represents an alternate gateway into the cannabinoid space as a substrate either to non-canonical cannabinoid synthase homologs or to synthetic chemical reactions. Herein, we describe the identification and repurposing of aromatic prenyltransferase (AtaPT), which when coupled with native enzymes of C. sativa can form an Escherichia coli production system for CBGA in cell lysates and CBG in whole cells. Engineering of AtaPT, guided by structural analysis, was performed to enhance its kinetics toward CBGA production for subsequent use in a proof-of-concept lysate system. For the first time, we show a synthetic biology platform for CBG biosynthesis in E. coli cells by employing AtaPT under an optimized microbial system. Our results have therefore set the foundation for sustainable production of well-researched and rarer cannabinoids in an E. coli chassis. Graphical Abstract [ABSTRACT FROM AUTHOR]

Photoenzymes use light to initiate biochemical reactions. Although rarely found in nature, their study has advanced understanding of how light energy can be harnessed to facilitate enzyme catalysis, which is also of importance to the design and engineering of man-made photocatalysts. Natural photoenzymes can be assigned to one of two families, based broadly on the nature of the light-sensing chromophores used, those being chlorophyll-like tetrapyrroles or flavins. In all cases, light absorption leads to excited state electron transfer, which in turn initiates photocatalysis. Reviewed here are recent findings relating to the structures and mechanisms of known photoenzymes. We highlight recent advances that have deepened understanding of mechanisms in biological photocatalysis. [ABSTRACT FROM AUTHOR]

Protochlorophyllide oxidoreductase (POR) catalyzes the light-driven reduction of protochlorophyllide (Pchlide), an essential, regulatory step in chlorophyll biosynthesis. The unique requirement of the enzyme for light has provided the opportunity to investigate how light energy can be harnessed to power biological catalysis and enzyme dynamics. Excited state interactions between the Pchlide molecule and the protein are known to drive the subsequent reaction chemistry. However, the structural features of POR and active site residues that are important for photochemistry and catalysis are currently unknown, because there is no crystal structure for POR. Here, we have used static and time-resolved spectroscopic measurements of a number of active site variants to study the role of a number of residues, which are located in the proposed NADPH/Pchlide binding site based on previous homology models, in the reaction mechanism of POR. Our findings, which are interpreted in the context of a new improved structural model, have identified several residues that are predicted to interact with the coenzyme or substrate. Several of the POR variants have a profound effect on the photochemistry, suggesting that multiple residues are important in stabilizing the excited state required for catalysis. Our work offers insight into how the POR active site geometry is finely tuned by multiple active site residues to support enzyme-mediated photochemistry and reduction of Pchlide, both of which are crucial to the existence of life on Earth. [ABSTRACT FROM AUTHOR]

Protein domain dynamics and electron transfer chemistry are often associated, but real-time analysis of domain motion in enzyme-catalysed reactions and the elucidation of mechanistic schemes that relate these motions to the reaction chemistry are major challenges for biological catalysis research. Previously we suggested that reduction of human cytochrome P450 reductase with the reducing coenzyme NADPH is accompanied by major structural re-orientation of the FMN- and FAD-binding domains through an inferred dynamic cycle of 'open' and 'closed' conformations of the enzyme (PLoS Biol, 2011, e1001222). However, these studies were restricted to stopped-flow/FRET analysis of the reductive half-reaction, and were compromised by fluorescence quenching of the acceptor by the flavin cofactors. Here we have improved the design of the FRET system, by using dye pairs with near-IR fluorescence, and extended studies on human cytochrome P450 reductase to the oxidative half-reaction using a double-mixing stopped-flow assay, thereby analysing in real-time conformational dynamics throughout the complete catalytic cycle. We correlate redox changes accompanying the reaction chemistry with protein dynamic changes observed by FRET, and show that redox chemistry drives a major re-orientation of the protein domains in both the reductive and oxidative half-reactions. Our studies using the tractable (soluble) surrogate electron acceptor cytochrome c provide a framework for analysing mechanisms of electron transfer in the endoplasmic reticulum between cytochrome P450 reductase and cognate P450 enzymes. More generally, our work emphasizes the importance of protein dynamics in intra- and inter-protein electron transfer, and establishes methodology for real-time analysis of structural changes throughout the catalytic cycle of complex redox proteins. [ABSTRACT FROM AUTHOR]

[Display omitted] • EPS genes were deleted from six Halomonas bluephagenesis strains for benefits of PHA synthesis. • EPS-KO cells demonstrate rapid self-flocculation and cell-lysis for reduced DSP cost. • EPS-KO induces remarkable cell filamentation, controllable under specific media regimes. • Successful PHA production in 5000 L FlipFlow bioreactor validates EPS-KO reduces DSP costs. The halophile Halomonas bluephagenesis has established itself as a cost-effective chassis in industrial bioproduction. However, downstream processing (DSP) expense is a persistent challenge. This study uses genetic knockout of exopolysaccharides (EPS-KO) in six industrially relevant H. bluephagenesis strains, each producing distinct polyhydroxyalkanoate (PHA), to evaluate their impact on reducing DSP costs. Remarkably, EPS-KO cells exhibited an unprecedented filamentous morphology, reaching lengths over 50 times (10–75 μm) that of standard cells. This unexpected behavior initially posed a challenge to industrial applicability due to rapid cell lysis under fermentation agitation. Systematic media supplementation of salt, carbon source, nitrogen, and essential minerals significantly influenced morphology, and tailored nutrient schemes that mitigated filamentation while maintaining production levels were designed. Scaled production in a 5000 L bioreactor demonstrated 85 % PHA and 96 g/L cell dry weight after 40 h fermentation, consistent with non-KO cells. During fermentation, EPS-KO enhanced cell-wall penetrability, reducing the need for high-cost γ-butyrolactone by 40 %. Additionally, a new innovative 5000 L FlipFlow bioreactor design cut rotation speed by 50 % and air input by 25 %. During DSP, centrifugation time decreased tenfold due to rapid cell self-flocculation (15–30 min), and the water and enzyme required for PHA purification was reduced by 30 % and 35 %, respectively. This study highlights the use of EPS-KO cells, a controlled media strategy, and low shear force FlipFlow bioreactor to successfully reduce DSP costs without sacrificing productivity. These novel findings represent a notable advancement in cost-saving strategies while presenting the intriguing discovery of media-controlled filamentous cell behavior. [ABSTRACT FROM AUTHOR]

Many enzymes utilize redox-coupled centers for performing catalysis where these centers are used to control and regulate the transfer of electrons required for catalysis, whose untimely delivery can lead to a state incapable of binding the substrate, i.e., a dead-end enzyme. Copper nitrite reductases (CuNiRs), which catalyze the reduction of nitrite to nitric oxide (NO), have proven to be a good model system for studying these complex processes including proton-coupled electron transfer (ET) and their orchestration for substrate binding/utilization. Recently, a two-domain CuNiR from a Rhizobia species (Br2DNiR) has been discovered with a substantially lower enzymatic activity where the catalytic type-2 Cu (T2Cu) site is occupied by two water molecules requiring their displacement for the substrate nitrite to bind. Single crystal spectroscopy combined with MSOX (multiple structures from one crystal) for both the as-isolated and nitrite-soaked crystals clearly demonstrate that inter-Cu ET within the coupled T1CuT2Cu redox system is heavily gated. Laser-flash photolysis and optical spectroscopy showed rapid ET from photoexcited NADH to the T1Cu center but little or no interCu ET in the absence of nitrite. Furthermore, incomplete reoxidation of the T1Cu site (∼20% electrons transferred) was observed in the presence of nitrite, consistent with a slow formation of NO species in the serial structures of the MSOX movie obtained from the nitrite-soaked crystal, which is likely to be responsible for the lower activity of this CuNiR. Our approach is of direct relevance for studying redox reactions in a wide range of biological systems including metalloproteins that make up at least 30% of all proteins. [ABSTRACT FROM AUTHOR]

α-Chiral amines are key intermediates for the synthesis of a plethora of chemical compounds at industrial scale.We present a biocatalytic hydrogen-borrowing amination of primary and secondary alcohols that allows for the efficient and environmentally benign production of enantiopure amines.The method relies on a combination of two enzymes: an alcohol dehydrogenase (from Aromatoleum sp., Lactobacillus sp., or Bacillus sp.) operating in tandem with an amine dehydrogenase (engineered from Bacillus sp.) to aminate a structurally diverse range of aromatic and aliphatic alcohols, yielding up to 96% conversion and 99% enantiomeric excess. Primary alcohols were aminated with high conversion (up to 99%). This redox self-sufficient cascade possesses high atom efficiency, sourcing nitrogen from ammonium and generating water as the sole by-product. [ABSTRACT FROM AUTHOR]