Unusual aldehyde reductase activity for the production of full-length fatty alcohol by cyanobacterial aldehyde deformylating oxygenase.

Aldehyde-deformylating oxygenase (ADO) is a non-heme di-iron enzyme that catalyzes the deformylation of aldehydes to generate alkanes/alkenes. In this study, we report for the first time that under anaerobic or limited oxygen conditions, Prochlorococcus marinus (PmADO) can generate full-length fatty alcohols from fatty aldehydes without eliminating a carbon unit. In contrast to ADO's native activity, which requires electrons from the Fd/FNR electron transfer complex, ADO's aldehyde reduction activity requires only NAD(P)H. Our results demonstrated that the yield of alcohol products could be affected by oxygen concentration and the type of aldehyde. Under strictly anaerobic conditions, yields of octanol were up to 31%. Moreover, metal cofactors are not involved in the aldehyde reductase activity of PmADO because the yields of alcohols obtained from apoenzyme and holoenzyme treated with various metals were similar under anaerobic conditions. In addition, PmADO prefers medium-chain aldehydes, specifically octanal (kcat/Km around 15 × 10-3 µM-1min-1). The findings herein highlight a new activity of PmADO, which may be applied as a biocatalyst for the industrial synthesis of fatty alcohols.

Enzymes, In Vivo Biocatalysis, and Metabolic Engineering for Enabling a Circular Economy and Sustainability.

Since the industrial revolution, the rapid growth and development of global industries have depended largely upon the utilization of coal-derived chemicals, and more recently, the utilization of petroleum-based chemicals. These developments have followed a linear economy model (produce, consume, and dispose). As the world is facing a serious threat from the climate change crisis, a more sustainable solution for manufacturing, i.e., circular economy in which waste from the same or different industries can be used as feedstocks or resources for production offers an attractive industrial/business model. In nature, biological systems, i.e., microorganisms routinely use their enzymes and metabolic pathways to convert organic and inorganic wastes to synthesize biochemicals and energy required for their growth. Therefore, an understanding of how selected enzymes convert biobased feedstocks into special (bio)chemicals serves as an important basis from which to build on for applications in biocatalysis, metabolic engineering, and synthetic biology to enable biobased processes that are greener and cleaner for the environment. This review article highlights the current state of knowledge regarding the enzymatic reactions used in converting biobased wastes (lignocellulosic biomass, sugar, phenolic acid, triglyceride, fatty acid, and glycerol) and greenhouse gases (CO2 and CH4) into value-added products and discusses the current progress made in their metabolic engineering. The commercial aspects and life cycle assessment of products from enzymatic and metabolic engineering are also discussed. Continued development in the field of metabolic engineering would offer diversified solutions which are sustainable and renewable for manufacturing valuable chemicals.

Luciferin Synthesis and Pesticide Detection by Luminescence Enzymatic Cascades.

D-Luciferin (D-LH2 ), a substrate of firefly luciferase (Fluc), is important for a wide range of bioluminescence applications. This work reports a new and green method using enzymatic reactions (HELP, HadA Enzyme for Luciferin Preparation) to convert 19 phenolic derivatives to 8 D-LH2 analogues with ≈51 % yield. The method can synthesize the novel 5'-methyl-D-LH2 and 4',5'-dimethyl-D-LH2 , which have never been synthesized or found in nature. 5'-Methyl-D-LH2 emits brighter and longer wavelength light than the D-LH2 . Using HELP, we further developed LUMOS (Luminescence Measurement of Organophosphate and Derivatives) technology for in situ detection of organophosphate pesticides (OPs) including parathion, methyl parathion, EPN, profenofos, and fenitrothion by coupling the reactions of OPs hydrolase and Fluc. The LUMOS technology can detect these OPs at parts per trillion (ppt) levels. The method can directly detect OPs in food and biological samples without requiring sample pretreatment.

Addition of formate dehydrogenase increases the production of renewable alkane from an engineered metabolic pathway.

An engineered metabolic pathway consisting of reactions that convert fatty acids to aldehydes and eventually alkanes would provide a means to produce biofuels from renewable energy sources. The enzyme aldehyde-deformylating oxygenase (ADO) catalyzes the conversion of aldehydes and oxygen to alkanes and formic acid and uses oxygen and a cellular reductant such as ferredoxin (Fd) as co-substrates. In this report, we aimed to increase ADO-mediated alkane production by converting an unused by-product, formate, to a reductant that can be used by ADO. We achieved this by including the gene (fdh), encoding formate dehydrogenase from Xanthobacter sp. 91 (XaFDH), into a metabolic pathway expressed in Escherichia coli Using this approach, we could increase bacterial alkane production, resulting in a conversion yield of ∼50%, the highest yield reported to date. Measuring intracellular nicotinamide concentrations, we found that E. coli cells harboring XaFDH have a significantly higher concentration of NADH and a higher NADH/NAD+ ratio than E. coli cells lacking XaFDH. In vitro analysis disclosed that ferredoxin (flavodoxin):NADP+ oxidoreductase could use NADH to reduce Fd and thus facilitate ADO-mediated alkane production. As formic acid can decrease the cellular pH, the addition of formate dehydrogenase could also maintain the cellular pH in the neutral range, which is more suitable for alkane production. We conclude that this simple, dual-pronged approach of increasing NAD(P)H and removing extra formic acid is efficient for increasing the production of renewable alkanes via synthetic biology-based approaches.

A Minimized Chemoenzymatic Cascade for Bacterial Luciferase in Bioreporter Applications.

Bacterial luciferase (Lux) catalyzes a bioluminescence reaction by using long-chain aldehyde, reduced flavin and molecular oxygen as substrates. The reaction can be applied in reporter gene systems for biomolecular detection in both prokaryotic and eukaryotic organisms. Because reduced flavin is unstable under aerobic conditions, another enzyme, flavin reductase, is needed to supply reduced flavin to the Lux-catalyzed reaction. To create a minimized cascade for Lux that would have greater ease of use, a chemoenzymatic reaction with a biomimetic nicotinamide (BNAH) was used in place of the flavin reductase reaction in the Lux system. The results showed that the minimized cascade reaction can be applied to monitor bioluminescence of the Lux reporter in eukaryotic cells effectively, and that it can achieve higher efficiencies than the system with flavin reductase. This development is useful for future applications as high-throughput detection tools for drug screening applications.

Creating Flavin Reductase Variants with Thermostable and Solvent-Tolerant Properties by Rational-Design Engineering.

We have employed computational approaches-FireProt and FRESCO-to predict thermostable variants of the reductase component (C1 ) of (4-hydroxyphenyl)acetate 3-hydroxylase. With the additional aid of experimental results, two C1 variants, A166L and A58P, were identified as thermotolerant enzymes, with thermostability improvements of 2.6-5.6 °C and increased catalytic efficiency of 2- to 3.5-fold. After heat treatment at 45 °C, both of the thermostable C1 variants remain active and generate reduced flavin mononucleotide (FMNH- ) for reactions catalyzed by bacterial luciferase and by the monooxygenase C2 more efficiently than the wild type (WT). In addition to thermotolerance, the A166L and A58P variants also exhibited solvent tolerance. Molecular dynamics (MD) simulations (6 ns) at 300-500 K indicated that mutation of A166 to L and of A58 to P resulted in structural changes with increased stabilization of hydrophobic interactions, and thus in improved thermostability. Our findings demonstrated that improvements in the thermostability of C1 enzyme can lead to broad-spectrum uses of C1 as a redox biocatalyst for future industrial applications.

The Mechanism of Sugar C-H Bond Oxidation by a Flavoprotein Oxidase Occurs by a Hydride Transfer Before Proton Abstraction.

Understanding the reaction mechanism underlying the functionalization of C-H bonds by an enzymatic process is one of the most challenging issues in catalysis. Here, combined approaches using density functional theory (DFT) analysis and transient kinetics were employed to investigate the reaction mechanism of C-H bond oxidation in d-glucose, catalyzed by the enzyme pyranose 2-oxidase (P2O). Unlike the mechanisms that have been conventionally proposed, our findings show that the first step of the C-H bond oxidation reaction is a hydride transfer from the C2 position of d-glucose to N5 of the flavin to generate a protonated ketone sugar intermediate. The proton is then transferred from the protonated ketone intermediate to a conserved residue, His548. The results show for the first time how specific interactions around the sugar binding site promote the hydride transfer and formation of the protonated ketone intermediate. The DFT results are also consistent with experimental results including the enthalpy of activation obtained from Eyring plots, as well as the results of kinetic isotope effect and site-directed mutagenesis studies. The mechanistic model obtained from this work may also be relevant to other reactions of various flavoenzyme oxidases that are generally used as biocatalysts in biotechnology applications.

One-Pot Bioconversion of l-Arabinose to l-Ribulose in an Enzymatic Cascade.

This work reports the one-pot enzymatic cascade that completely converts l-arabinose to l-ribulose using four reactions catalyzed by pyranose 2-oxidase (P2O), xylose reductase, formate dehydrogenase, and catalase. As wild-type P2O is specific for the oxidation of six-carbon sugars, a pool of P2O variants was generated based on rational design to change the specificity of the enzyme towards the oxidation of l-arabinose at the C2-position. The variant T169G was identified as the best candidate, and this had an approximately 40-fold higher rate constant for the flavin reduction (sugar oxidation) step, as compared to the wild-type enzyme. Computational calculations using quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) showed that this improvement is due to a decrease in the steric effects at the axial C4-OH of l-arabinose, which allows a reduction in the distance between the C2-H and flavin N5, facilitating hydride transfer and enabling flavin reduction.

The reaction mechanism of methyl-coenzyme M reductase: how an enzyme enforces strict binding order.

Methyl-coenzyme M reductase (MCR) is a nickel tetrahydrocorphinoid (coenzyme F430) containing enzyme involved in the biological synthesis and anaerobic oxidation of methane. MCR catalyzes the conversion of methyl-2-mercaptoethanesulfonate (methyl-SCoM) and N-7-mercaptoheptanoylthreonine phosphate (CoB7SH) to CH4 and the mixed disulfide CoBS-SCoM. In this study, the reaction of MCR from Methanothermobacter marburgensis, with its native substrates was investigated using static binding, chemical quench, and stopped-flow techniques. Rate constants were measured for each step in this strictly ordered ternary complex catalytic mechanism. Surprisingly, in the absence of the other substrate, MCR can bind either substrate; however, only one binary complex (MCR·methyl-SCoM) is productive whereas the other (MCR·CoB7SH) is inhibitory. Moreover, the kinetic data demonstrate that binding of methyl-SCoM to the inhibitory MCR·CoB7SH complex is highly disfavored (Kd = 56 mM). However, binding of CoB7SH to the productive MCR·methyl-SCoM complex to form the active ternary complex (CoB7SH·MCR(Ni(I))·CH3SCoM) is highly favored (Kd = 79 µM). Only then can the chemical reaction occur (kobs = 20 s(-1) at 25 °C), leading to rapid formation and dissociation of CH4 leaving the binary product complex (MCR(Ni(II))·CoB7S(-)·SCoM), which undergoes electron transfer to regenerate Ni(I) and the final product CoBS-SCoM. This first rapid kinetics study of MCR with its natural substrates describes how an enzyme can enforce a strictly ordered ternary complex mechanism and serves as a template for identification of the reaction intermediates.

Proton-coupled electron transfer and adduct configuration are important for C4a-hydroperoxyflavin formation and stabilization in a flavoenzyme.

Determination of the mechanism of dioxygen activation by flavoenzymes remains one of the most challenging problems in flavoenzymology for which the underlying theoretical basis is not well understood. Here, the reaction of reduced flavin and dioxygen catalyzed by pyranose 2-oxidase (P2O), a flavoenzyme oxidase that is unique in its formation of C4a-hydroperoxyflavin, was investigated by density functional calculations, transient kinetics, and site-directed mutagenesis. Based on work from the 1970s-1980s, the current understanding of the dioxygen activation process in flavoenzymes is believed to involve electron transfer from flavin to dioxygen and subsequent proton transfer to form C4a-hydroperoxyflavin. Our findings suggest that the first step of the P2O reaction is a single electron transfer coupled with a proton transfer from the conserved residue, His548. In fact, proton transfer enhances the electron acceptor ability of dioxygen. The resulting ·OOH of the open-shell diradical pair is placed in an optimal position for the formation of C4a-hydroperoxyflavin. Furthermore, the C4a-hydroperoxyflavin is stabilized by the side chains of Thr169, His548, and Asn593 in a "face-on" configuration where it can undergo a unimolecular reaction to generate H2O2 and oxidized flavin. The computational results are consistent with kinetic studies of variant forms of P2O altered at residues Thr169, His548, and Asn593, and kinetic isotope effects and pH-dependence studies of the wild-type enzyme. In addition, the calculated energy barrier is in agreement with the experimental enthalpy barrier obtained from Eyring plots. This work revealed new insights into the reaction of reduced flavin with dioxygen, demonstrating that the positively charged residue (His548) plays a significant role in catalysis by providing a proton for a proton-coupled electron transfer in dioxygen activation. The interaction around the N5-position of the C4a-hydroperoxyflavin is important for dictating the stability of the intermediate.

Nanocrystalline BEA-CNT Composites with High Metal Dispersion Obtained via Inter-Zeolite Transformation for Antibacterial Application.

The rational design of interface materials containing carbon nanotubes (CNTs) and zeolites (zeolite-CNTs) is a promising perspective in chemical and biochemical communities because they exhibit several outstanding properties such as tunable hydrophobicity-hydrophilicity at interfaces. In this contribution, we report the fabrication of Ag-incorporated nanocrystalline BEA-carbon nanotube (CNT) composites via the one-pot inter-zeolite transformation of the micron-sized FAU-CNT composite in the presence of a Ag precursor. By varying the crystallization time, the inter-zeolite transformation mechanism was explored. Indeed, this process involves an amorphous intermediate of aluminosilicate species with a significant change of the crystal morphology in the presence of CNTs in the synthesis gel. Interestingly, the redispersion of metal particles was observed after the inter-zeolite transformation process, resulting in the high dispersion of metal nanoparticles over BEA nanocrystals. Notably, it was revealed that the Ag sites were also stabilized in the presence of CNT interfaces, leading to the availability of highly active Ag+ ions. To illustrate the beneficial aspect of designer materials, the synthesized Ag-incorporated BEA-CNT composites exhibited high antibacterial activity againstEscherichia coli due to the synergistic effect of the active Ag+ species and appropriate hydrophobic and hydrophilic properties of the hybrid material interfaces. This first example opens up perspectives of the rational design of zeolite-CNT interfaces with high metal dispersion via the inter-zeolite transformation approach for biomedical applications.

Reduction kinetics of 3-hydroxybenzoate 6-hydroxylase from Rhodococcus jostii RHA1.

3-Hydroxybenzoate 6-hydroxylase (3HB6H) from Rhodococcus jostii RHA1 is a nicotinamide adenine dinucleotide (NADH)-specific flavoprotein monooxygenase involved in microbial aromatic degradation. The enzyme catalyzes the para hydroxylation of 3-hydroxybenzoate (3-HB) to 2,5-dihydroxybenzoate (2,5-DHB), the ring-fission fuel of the gentisate pathway. In this study, the kinetics of reduction of the enzyme-bound flavin by NADH was investigated at pH 8.0 using a stopped-flow spectrophotometer, and the data were analyzed comprehensively according to kinetic derivations and simulations. Observed rate constants for reduction of the free enzyme by NADH under anaerobic conditions were linearly dependent on NADH concentrations, consistent with a one-step irreversible reduction model with a bimolecular rate constant of 43 ± 2 M(-1) s(-1). In the presence of 3-HB, observed rate constants for flavin reduction were hyperbolically dependent on NADH concentrations and approached a limiting value of 48 ± 2 s(-1). At saturating concentrations of NADH (10 mM) and 3-HB (10 mM), the reduction rate constant is ~51 s(-1), whereas without 3-HB, the rate constant is 0.43 s(-1) at a similar NADH concentration. A similar stimulation of flavin reduction was found for the enzyme-product (2,5-DHB) complex, with a rate constant of 45 ± 2 s(-1). The rate enhancement induced by aromatic ligands is not due to a thermodynamic driving force because Em 0 for the enzyme-substrate complex is -179 ± 1 mV compared to an E(m)(0) of -175 ± 2 mV for the free enzyme. It is proposed that the reduction mechanism of 3HB6H involves an isomerization of the initial enzyme-ligand complex to a fully activated form before flavin reduction takes place.

Hydrogen peroxide elimination from C4a-hydroperoxyflavin in a flavoprotein oxidase occurs through a single proton transfer from flavin N5 to a peroxide leaving group.

C4a-hydroperoxyflavin is found commonly in the reactions of flavin-dependent monooxygenases, in which it plays a key role as an intermediate that incorporates an oxygen atom into substrates. Only recently has evidence for its involvement in the reactions of flavoprotein oxidases been reported. Previous studies of pyranose 2-oxidase (P2O), an enzyme catalyzing the oxidation of pyranoses using oxygen as an electron acceptor to generate oxidized sugars and hydrogen peroxide (H(2)O(2)), have shown that C4a-hydroperoxyflavin forms in P2O reactions before it eliminates H(2)O(2) as a product (Sucharitakul, J., Prongjit, M., Haltrich, D., and Chaiyen, P. (2008) Biochemistry 47, 8485-8490). In this report, the solvent kinetic isotope effects (SKIE) on the reaction of reduced P2O with oxygen were investigated using transient kinetics. Our results showed that D(2)O has a negligible effect on the formation of C4a-hydroperoxyflavin. The ensuing step of H(2)O(2) elimination from C4a-hydroperoxyflavin was shown to be modulated by an SKIE of 2.8 ± 0.2, and a proton inventory analysis of this step indicates a linear plot. These data suggest that a single-proton transfer process causes SKIE at the H(2)O(2) elimination step. Double and single mixing stopped-flow experiments performed in H(2)O buffer revealed that reduced flavin specifically labeled with deuterium at the flavin N5 position generated kinetic isotope effects similar to those found with experiments performed with the enzyme pre-equilibrated in D(2)O buffer. This suggests that the proton at the flavin N5 position is responsible for the SKIE and is the proton-in-flight that is transferred during the transition state. The mechanism of H(2)O(2) elimination from C4a-hydroperoxyflavin is consistent with a single proton transfer from the flavin N5 to the peroxide leaving group, possibly via the formation of an intramolecular hydrogen bridge.

Rational-Design Engineering to Improve Enzyme Thermostability.

The fundamentals of thermostability engineering need to be carried out for proteins with low thermal stability to expand their utilization. Thus, comprehension of the thermal stability regulating factors of proteins is needful for the engineering of their thermostability. Protein engineering aims to overcome their natural limitations in tough conditions by refining protein stability and activity. Rational-design approach requires a crystal structure dataset along with the biophysical information, protein function, and sequence-based data, especially consensus sequence that is favorable for the protein folding during natural evolution. It can be attained by either single- or multiple-point mutation, by which amino acids are changed. In fact, these mutation approaches show several benefits. For example, the offered mutations are produced after an evaluation and design, which raise the chance to acquire favorable mutations. The rational-design engineering can improve the biochemical properties of enzymes, including the kinetic behaviors, substrate specificity, thermostability, and organic solvent tolerance. Moreover, this approach considerably reduces the library size, so less effort and time can be employed. Here, we apply the computational algorithms and programs with experiments to create thermostable enzymes that will be beneficial for future applications.

Detection of cellular metabolites by redox enzymatic cascades.

Detection of cellular metabolites that are disease biomarkers is important for human healthcare monitoring and assessing prognosis and therapeutic response. Accurate and rapid detection of microbial metabolites and pathway intermediates is also crucial for the process optimization required for development of bioconversion methods using metabolically engineered cells. Various redox enzymes can generate electrons that can be employed in enzyme-based biosensors and in the detection of cellular metabolites. These reactions can directly transform target compounds into various readout signals. By incorporating engineered enzymes into enzymatic cascades, the readout signals can be improved in terms of accuracy and sensitivity. This review critically discusses selected redox enzymatic and chemoenzymatic cascades currently employed for detection of human- and microbe-related cellular metabolites including, amino acids, d-glucose, inorganic ions (pyrophosphate, phosphate, and sulfate), nitro- and halogenated phenols, NAD(P)H, fatty acids, fatty aldehyde, alkane, short chain acids, and cellular metabolites.

Identification of a catalytic base for sugar oxidation in the pyranose 2-oxidase reaction.

Pyranose 2-oxidase (P2O) catalyzes the oxidation of aldopyranoses to form 2-keto sugars and H(2)O(2) . In this study, the mechanistic role of the conserved residues His548 and Asn593 in P2O was investigated by using site-directed mutagenesis, transient kinetics, and pH-dependence studies. As single mutants of H548 resulted in mixed populations of noncovalently bound and covalently linked FAD, double mutants containing H167A were constructed, in which the covalent histidyl-FAD linkage was removed in addition to having the H548 mutation. Single mutants H548A, H548N, H548S, H548D and double mutants (with H167A) could not be reduced by D-glucose. For the H167A/H548R mutant, the flavin could be reduced by D-glucose with the reduction rate constant about 220 times lower than that of the H167A mutant. The pH-dependence studies of H167A/H548R indicated that the rate constant of flavin reduction increased about 360-fold upon a pH rise corresponding to pK(a) >10.1, whereas the reactions of the wild-type and H167A mutant enzymes were pH independent. Therefore, the data suggest that a pK(a) value of >10.1 in the mutant enzyme is associated with the Arg548 residue, and that this residue must be unprotonated to efficiently catalyze flavin reduction. The data imply that for the wild-type P2O, the conserved His548 should be unprotonated in the pH range studied. The unprotonated His548 can act as a general base to abstract the 2-hydroxyl proton of D-glucose and initiate hydride transfer from the substrate to the flavin. Studies of the single mutant N593H showed that the flavin reduction rate constant was 114 times lower than that of the wild-type enzyme and was pH independent, while the K(d) for D-glucose binding was 19 times greater.

Modulations of the reduction potentials of flavin-based electron bifurcation complexes and semiquinone stabilities are key to control directional electron flow.

The flavin-based electron bifurcation (FBEB) system from Acidaminococcus fermentans is composed of the electron transfer flavoprotein (EtfAB) and butyryl-CoA dehydrogenase (Bcd). α-FAD binds to domain II of the A-subunit of EtfAB, ß-FAD to the B-subunit of EtfAB and δ-FAD to Bcd. NADH reduces ß-FAD to ß-FADH- , which bifurcates one electron to the high potential α-FAD•- semiquinone followed by the other to the low potential ferredoxin (Fd). As deduced from crystal structures, upon interaction of EtfAB with Bcd, the formed α-FADH- approaches δ-FAD by rotation of domain II, yielding δ-FAD•- . Repetition of this process leads to a second reduced ferredoxin (Fd- ) and δ-FADH- , which reduces crotonyl-CoA to butyryl-CoA. In this study, we measured the redox properties of the components EtfAB, EtfaB (Etf without α-FAD), Bcd, and Fd, as well as of the complexes EtfaB:Bcd, EtfAB:Bcd, EtfaB:Fd, and EftAB:Fd. In agreement with the structural studies, we have shown for the first time that the interaction of EtfAB with Bcd drastically decreases the midpoint reduction potential of α-FAD to be within the same range of that of ß-FAD and to destabilize the semiquinone of α-FAD. This finding clearly explains that these interactions facilitate the passing of electrons from ß-FADH- via α-FAD•- to the final electron acceptor δ-FAD•- on Bcd. The interactions modulate the semiquinone stability of δ-FAD in an opposite way by having a greater semiquinone stability than in free Bcd.

Kinetic isotope effects on the noncovalent flavin mutant protein of pyranose 2-oxidase reveal insights into the flavin reduction mechanism.

Pyranose 2-oxidase (P2O) from Trametes multicolor contains a flavin adenine dinucleotide (FAD) cofactor covalently linked to the N(3) atom of His167. The enzyme catalyzes the oxidation of aldopyranoses by molecular oxygen to generate 2-keto-aldoses and H(2)O(2) as products. In this study, the transient kinetics and primary and solvent kinetic isotope effects of the mutant in which His167 has been replaced with Ala (H167A) were investigated, to elucidate the functional role of the 8a-N(3)-histidyl FAD linkage and to gain insights into the reaction mechanism of P2O. The results indicate that the covalent linkage is mainly important for a reductive half-reaction in which the FAD cofactor is reduced by d-glucose, while it is not important for an oxidative half-reaction in which oxygen reacts with the reduced FAD to generate H(2)O(2). d-Glucose binds to H167A via multiple binding modes before the formation of the active Michaelis complex, and the rate constant of flavin reduction decreases approximately 22-fold compared to that of the wild-type enzyme. The reduction of H167A using d-glucose isotopes (2-d-d-glucose, 3-d-d-glucose, and 1,2,3,4,5,6,6-d(7)-d-glucose) as substrates indicates that the primary isotope effect results only from substitution at the C2 position, implying that H167A catalyzes the oxidation of d-glucose regiospecifically at this position. No solvent kinetic isotope effect was detected during the reductive half-reaction of the wild-type or H167A enzyme, implying that the deprotonation of the d-glucose C2-OH group may occur readily upon the binding to P2O and is not synchronized with the cleavage of the d-glucose C2-H bond. The mutation has no drastic effect on the oxidative half-reaction of P2O, as H167A is very similar to the wild-type enzyme with respect to the kinetic constants and the formation of the C4a-hydroperoxyflavin intermediate. Kinetic mechanisms for both half-reactions of H167A were proposed on the basis of transient kinetic data and were verified by kinetic simulations and steady-state kinetic parameters.

Microbial degradation of halogenated aromatics: molecular mechanisms and enzymatic reactions.

Halogenated aromatics are used widely in various industrial, agricultural and household applications. However, due to their stability, most of these compounds persist for a long time, leading to accumulation in the environment. Biological degradation of halogenated aromatics provides sustainable, low-cost and environmentally friendly technologies for removing these toxicants from the environment. This minireview discusses the molecular mechanisms of the enzymatic reactions for degrading halogenated aromatics which naturally occur in various microorganisms. In general, the biodegradation process (especially for aerobic degradation) can be divided into three main steps: upper, middle and lower metabolic pathways which successively convert the toxic halogenated aromatics to common metabolites in cells. The most difficult step in the degradation of halogenated aromatics is the dehalogenation step in the middle pathway. Although a variety of enzymes are involved in the degradation of halogenated aromatics, these various pathways all share the common feature of eventually generating metabolites for utilizing in the energy-producing metabolic pathways in cells. An in-depth understanding of how microbes employ various enzymes in biodegradation can lead to the development of new biotechnologies via enzyme/cell/metabolic engineering or synthetic biology for sustainable biodegradation processes.

Structure and function relationships of sugar oxidases and their potential use in biocatalysis.

Several sugar oxidases that catalyze the oxidation of sugars have been isolated and characterized. These enzymes can be classified as flavoenzyme due to the presence of flavin adenine dinucleotide (FAD) as a cofactor. Sugar oxidases have been proposed to be the key biocatalyst in biotransformation of carbohydrates which can potentially convert sugars to provide a pool of intermediates for synthesis of rare sugars, fine chemicals and drugs. Moreover, sugar oxidases have been applied in biosensing of various biomolecules in food industries, diagnosis of diseases and environmental pollutant detection. This review provides the discussions on general properties, current mechanistic understanding, structural determination, biocatalytic application, and biosensor integration of representative sugar oxidase enzymes, namely pyranose 2-oxidase (P2O), glucose oxidase (GO), hexose oxidase (HO), and oligosaccharide oxidase. The information regarding the relationship between structure and function of these sugar oxidases points out the key properties of this particular group of enzymes that can be modified by engineering, which had resulted in a remarkable economic importance.