On the Mechanisms of Hypohalous Acid Formation and Electrophilic Halogenation by Non-Native Halogenases.

Enzymatic electrophilic halogenation is a mild tool for functionalization of diverse organic compounds. Only a few groups of native halogenases are capable of catalyzing such a reaction. In this study, we used a mechanism-guided strategy to discover the electrophilic halogenation activity catalyzed by non-native halogenases. As the ability to form a hypohalous acid (HOX) is key for halogenation, flavin-dependent monooxygenases/oxidases capable of forming C4a-hydroperoxyflavin (FlC4a-OOH), such as dehalogenase, hydroxylases, luciferase and pyranose-2-oxidase (P2O), and flavin reductase capable of forming H2O2 were explored for their abilities to generate HOX in situ. Transient kinetic analyses using stopped-flow spectrophotometry/fluorometry and product analysis indicate that FlC4a-OOH in dehalogenases, selected hydroxylases and luciferases, but not in P2O can form HOX; however, the HOX generated from FlC4a-OOH cannot halogenate their substrates. Remarkably, in situ H2O2 generated by P2O can form HOI and also iodinate various compounds. Because not all enzymes capable of forming FlC4a-OOH can react with halides to form HOX, QM/MM calculations, site-directed mutagenesis and structural analysis were carried out to elucidate the mechanism underlying HOX formation and characterize the active site environment. Our findings shed light on identifying new halogenase scaffolds besides the currently known enzymes and have invoked a new mode of chemoenzymatic halogenation.

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.

QM/MM Molecular Modeling Reveals Mechanism Insights into Flavin Peroxide Formation in Bacterial Luciferase.

Bacterial luciferase (Lux) catalyzes oxidation of reduced flavin mononucleotide (FMN) and aldehyde to form oxidized FMN and carboxylic acid via molecular oxygen with concomitant light generation. The enzyme is useful for various detection applications in biomedical experiments. Upon reacting with oxygen, the reduced FMN generates C4a-peroxy-FMN (FMNH-C4a-OO-) as a reactive intermediate, which is required for light generation. However, the mechanism and control of FMNH-C4a-OO- formation are not clear. This work investigated the reaction of FMNH-C4a-OO- formation in Lux using QM/MM methods. The B3LYP/6-31G*/CHARMM27 calculations indicate that Lux controls the formation of FMNH-C4a-OO- via the conserved His44 residue. The steps in intermediate formation are found to be as follows: (i) H+ reacts with O2 to generate +OOH. (ii) +OOH attacks C4a of FMNH- to generate FMNH-C4a-OOH. (iii) H+ is transferred from FMNH-C4a-OOH to His44 to generate FMNH-C4a-OO- while His44 stabilizes FMNH-C4a-OO- by forming a hydrogen bond to an oxygen atom. This controlling key mechanism for driving the change from FMNH-C4a-OOH to the FMNH-C4a-OO- adduct is confirmed because FMNH-C4a-OO- is more stable than FMNH-C4a-OOH in the luciferase active site.

Catalytic and structural insights into a stereospecific and thermostable Class II aldolase HpaI from Acinetobacter baumannii.

Aldolases catalyze the reversible reactions of aldol condensation and cleavage and have strong potential for the synthesis of chiral compounds, widely used in pharmaceuticals. Here, we investigated a new Class II metal aldolase from the p-hydroxyphenylacetate degradation pathway in Acinetobacter baumannii, 4-hydroxy-2-keto-heptane-1,7-dioate aldolase (AbHpaI), which has various properties suitable for biocatalysis, including stereoselectivity/stereospecificity, broad aldehyde utilization, thermostability, and solvent tolerance. Notably, the use of Zn2+ by AbHpaI as a native cofactor is distinct from other enzymes in this class. AbHpaI can also use other metal ion (M2+) cofactors, except Ca2+, for catalysis. We found that Zn2+ yielded the highest enzyme complex thermostability (Tm of 87 °C) and solvent tolerance. All AbHpaI•M2+ complexes demonstrated preferential cleavage of (4R)-2-keto-3-deoxy-D-galactonate ((4R)-KDGal) over (4S)-2-keto-3-deoxy-D-gluconate ((4S)-KDGlu), with AbHpaI•Zn2+ displaying the highest R/S stereoselectivity ratio (sixfold higher than other M2+ cofactors). For the aldol condensation reaction, AbHpaI•M2+ only specifically forms (4R)-KDGal and not (4S)-KDGlu and preferentially catalyzes condensation rather than cleavage by ∼40-fold. Based on 11 X-ray structures of AbHpaI complexed with M2+ and ligands at 1.85 to 2.0 Å resolution, the data clearly indicate that the M2+ cofactors form an octahedral geometry with Glu151 and Asp177, pyruvate, and water molecules. Moreover, Arg72 in the Zn2+-bound form governs the stereoselectivity/stereospecificity of AbHpaI. X-ray structures also show that Ca2+ binds at the trimer interface via interaction with Asp51. Hence, we conclude that AbHpaI•Zn2+ is distinctive from its homologues in substrate stereospecificity, preference for aldol formation over cleavage, and protein robustness, and is attractive for biocatalytic applications.

Dissecting the low catalytic capability of flavin-dependent halogenases.

Although flavin-dependent halogenases (FDHs) are attractive biocatalysts, their practical applications are limited because of their low catalytic efficiency. Here, we investigated the reaction mechanisms and structures of tryptophan 6-halogenase (Thal) from Streptomyces albogriseolus using stopped-flow, rapid-quench flow, quantum/mechanics molecular mechanics calculations, crystallography, and detection of intermediate (hypohalous acid [HOX]) liberation. We found that the key flavin intermediate, C4a-hydroperoxyflavin (C4aOOH-FAD), formed by Thal and other FDHs (tryptophan 7-halogenase [PrnA] and tryptophan 5-halogenase [PyrH]), can react with I-, Br-, and Cl- but not F- to form C4a-hydroxyflavin and HOX. Our experiments revealed that I- reacts with C4aOOH-FAD the fastest with the lowest energy barrier and have shown for the first time that a significant amount of the HOX formed leaks out as free HOX. This leakage is probably a major cause of low product coupling ratios in all FDHs. Site-saturation mutagenesis of Lys79 showed that changing Lys79 to any other amino acid resulted in an inactive enzyme. However, the levels of liberated HOX of these variants are all similar, implying that Lys79 probably does not form a chloramine or bromamine intermediate as previously proposed. Computational calculations revealed that Lys79 has an abnormally lower pKa compared with other Lys residues, implying that the catalytic Lys may act as a proton donor in catalysis. Analysis of new X-ray structures of Thal also explains why premixing of FDHs with reduced flavin adenine dinucleotide generally results in abolishment of C4aOOH-FAD formation. These findings reveal the hidden factors restricting FDHs capability which should be useful for future development of FDHs applications.

Protonation status and control mechanism of flavin-oxygen intermediates in the reaction of bacterial luciferase.

Bacterial luciferase catalyzes a bioluminescent reaction by oxidizing long-chain aldehydes to acids using reduced FMN and oxygen as co-substrates. Although a flavin C4a-peroxide anion is postulated to be the intermediate reacting with aldehyde prior to light liberation, no clear identification of the protonation status of this intermediate has been reported. Here, transient kinetics, pH variation, and site-directed mutagenesis were employed to probe the protonation state of the flavin C4a-hydroperoxide in bacterial luciferase. The first observed intermediate, with a λmax of 385 nm, transformed to an intermediate with a λmax of 375 nm. Spectra of the first observed intermediate were pH-dependent, with a λmax of 385 nm at pH < 8.5 and 375 at pH > 9, correlating with a pKa of 7.7-8.1. These data are consistent with the first observed flavin C4a intermediate at pH < 8.5 being the protonated flavin C4a-hydroperoxide, which loses a proton to become an active flavin C4a-peroxide. Stopped-flow studies of His44Ala, His44Asp, and His44Asn variants showed only a single intermediate with a λmax of 385 nm at all pH values, and none of these variants generate light. These data indicate that His44 variants only form a flavin C4a-hydroperoxide, but not an active flavin C4a-peroxide, indicating an essential role for His44 in deprotonating the flavin C4a-hydroperoxide and initiating chemical catalysis. We also investigated the function of the adjacent His45; stopped-flow data and molecular dynamics simulations identify the role of this residue in binding reduced FMN.

Mechanistic insights into the dual activities of the single active site of l-lysine oxidase/monooxygenase from Pseudomonas sp. AIU 813.

l-Lysine oxidase/monooxygenase (l-LOX/MOG) from Pseudomonas sp. AIU 813 catalyzes the mixed bioconversion of l-amino acids, particularly l-lysine, yielding an amide and carbon dioxide by an oxidative decarboxylation (i.e. apparent monooxygenation), as well as oxidative deamination (hydrolysis of oxidized product), resulting in α-keto acid, hydrogen peroxide (H2O2), and ammonia. Here, using high-resolution MS and monitoring transient reaction kinetics with stopped-flow spectrophotometry, we identified the products from the reactions of l-lysine and l-ornithine, indicating that besides decarboxylating imino acids (i.e. 5-aminopentanamide from l-lysine), l-LOX/MOG also decarboxylates keto acids (5-aminopentanoic acid from l-lysine and 4-aminobutanoic acid from l-ornithine). The reaction of reduced enzyme and oxygen generated an imino acid and H2O2, with no detectable C4a-hydroperoxyflavin. Single-turnover reactions in which l-LOX/MOG was first reduced by l-lysine to form imino acid before mixing with various compounds revealed that under anaerobic conditions, only hydrolysis products are present. Similar results were obtained upon H2O2 addition after enzyme denaturation. H2O2 addition to active l-LOX/MOG resulted in formation of more 5-aminopentanoic acid, but not 5-aminopentamide, suggesting that H2O2 generated from l-LOX/MOG in situ can result in decarboxylation of the imino acid, yielding an amide product, and extra H2O2 resulted in decarboxylation only of keto acids. Molecular dynamics simulations and detection of charge transfer species suggested that interactions between the substrate and its binding site on l-LOX/MOG are important for imino acid decarboxylation. Structural analysis indicated that the flavoenzyme oxidases catalyzing decarboxylation of an imino acid all share a common plug loop configuration that may facilitate this decarboxylation.

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.

Identification of a Hotspot Residue for Improving the Thermostability of a Flavin-Dependent Monooxygenase.

HadA is a flavin-dependent monooxygenase that can catalyze the denitration and dehalogenation of a wide variety of toxicants such as pesticides. Although these enzymatic reactions are useful for bioremediation or biocatalysis, the application of HadA for these purposes is not yet possible because of its low thermostability. In this work we have engineered HadA to be more thermostable through the use of structural, in silico, and rational approaches. The X-ray structure of HadA was solved to obtain a reliable three-dimensional protein model for further prediction of thermostable variants. In silico analysis by using two bioinformatic tools-FireProt and Disulfide by Design-suggested 102 variants that we then further refined by applying rational criteria including the location of a particular residue and its nearby interactions, as well as other biophysical parameters to narrow down the list to six candidates. The G513Y variant was found to be an optimal engineered candidate because it has significantly improved stability relative to the wild-type enzyme and equivalent activity. G513Y has an activity half-life 72 (50 °C) and 160 times (45 °C) longer than that of the wild-type enzyme. Coupled together with thermostable reactions of reduced flavin and NADH-regenerating systems, the G513Y variant can be used to catalyze denitration of 4nitrophenol at 45 °C. Structure/sequence alignments of HadA and its homologues indicate that several flavin-dependent monooxygenases also contain amino acid residues homologous to the G513 of HadA, hence opening up the possibility of applying this engineering approach to improving their thermostabilities as well. Molecular dynamics (MD) simulations confirmed that the improved thermostability of the G513Y variant was due to aromatic hydrocarbon interactions between Y513 and N359, L347, G348, and F349.

Projector-Based Embedding Eliminates Density Functional Dependence for QM/MM Calculations of Reactions in Enzymes and Solution.

Combined quantum mechanics/molecular mechanics (QM/MM) methods are increasingly widely utilized in studies of reactions in enzymes and other large systems. Here, we apply a range of QM/MM methods to investigate the Claisen rearrangement of chorismate to prephenate, in solution, and in the enzyme chorismate mutase. Using projector-based embedding in a QM/MM framework, we apply treatments up to the CCSD(T) level. We test a range of density functional QM/MM methods and QM region sizes. The results show that the calculated reaction energetics are significantly more sensitive to the choice of density functional than they are to the size of the QM region in these systems. Projector-based embedding of a wave function method in DFT reduced the 13 kcal/mol spread in barrier heights calculated at the DFT/MM level to a spread of just 0.3 kcal/mol, essentially eliminating dependence on the functional. Projector-based embedding of correlated ab initio methods provides a practical method for achieving high accuracy for energy profiles derived from DFT and DFT/MM calculations for reactions in condensed phases.

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 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.

QM/MM molecular modelling on mutation effect of chorismate synthase enzyme catalysis.

Chorismate synthase (CS) catalyzes the conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate which is a key intermediate in the biosynthesis of aromatic amino acids. CS enzyme is a new target for antibacterial drugs. Even though several reaction mechanisms have been proposed, the catalytic mechanism is still unclear. QM/MM adiabatic mapping calculations were performed in order to investigate roles of this enzyme. High-accuracy SCS-MP2/aVDZ/CHARMM27 calculations indicated that the reaction pathway has three steps; (i) proton transfer from reduced flavin mononucleotide (FMNH2) to D339, (ii) proton transfer from EPSP to FMNH- and (iii) phosphate elimination. Adiabatic mapping calculations indicated that H110 and R48 residues play essential catalyst roles for CS enzyme catalysis by transition state (TS) and product stabilizations via charge polarization and hydrogen bonding to EPSP and/or FMNH2. A high accuracy calculation - SCS-MP2/aVDZ/CHARMM27 method was employed to obtain the accurate reaction mechanism pathway and to evaluate the effect of amino acid residues in the active site on the enzyme catalysis. The potential energy barriers of the reactions of H110A and R48A were found to increase. The CS catalysis was consequently slowed down due to missing the TS and product stabilizations.

Improved scFv anti-HIV-1 p17 binding affinity guided from the theoretical calculation of pairwise decomposition energies and computational alanine scanning.

Computational approaches have been used to evaluate and define important residues for protein-protein interactions, especially antigen-antibody complexes. In our previous study, pairwise decomposition of residue interaction energies of single chain Fv with HIV-1 p17 epitope variants has indicated the key specific residues in the complementary determining regions (CDRs) of scFv anti-p17. In this present investigation in order to determine whether a specific side chain group of residue in CDRs plays an important role in bioactivity, computational alanine scanning has been applied. Molecular dynamics simulations were done with several complexes of original scFv anti-p17 and scFv anti-p17mutants with HIV-1 p17 epitope variants with a production run up to 10 ns. With the combination of pairwise decomposition residue interaction and alanine scanning calculations, the point mutation has been initially selected at the position MET100 to improve the residue binding affinity. The calculated docking interaction energy between a single mutation from methionine to either arginine or glycine has shown the improved binding affinity, contributed from the electrostatic interaction with the negative favorably interaction energy, compared to the wild type. Theoretical calculations agreed well with the results from the peptide ELISA results.

Analysis of chorismate mutase catalysis by QM/MM modelling of enzyme-catalysed and uncatalysed reactions.

Chorismate mutase is at the centre of current controversy about fundamental features of biological catalysts. Some recent studies have proposed that catalysis in this enzyme does not involve transition state (TS) stabilization but instead is due largely to the formation of a reactive conformation of the substrate. To understand the origins of catalysis, it is necessary to compare equivalent reactions in different environments. The pericyclic conversion of chorismate to prephenate catalysed by chorismate mutase also occurs (much more slowly) in aqueous solution. In this study we analyse the origins of catalysis by comparison of multiple quantum mechanics/molecular mechanics (QM/MM) reaction pathways at a reliable, well tested level of theory (B3LYP/6-31G(d)/CHARMM27) for the reaction (i) in Bacillus subtilis chorismate mutase (BsCM) and (ii) in aqueous solvent. The average calculated reaction (potential energy) barriers are 11.3 kcal mol(-1) in the enzyme and 17.4 kcal mol(-1) in water, both of which are in good agreement with experiment. Comparison of the two sets of reaction pathways shows that the reaction follows a slightly different reaction pathway in the enzyme than in it does in solution, because of a destabilization, or strain, of the substrate in the enzyme. The substrate strain energy within the enzyme remains constant throughout the reaction. There is no unique reactive conformation of the substrate common to both environments, and the transition state structures are also different in the enzyme and in water. Analysis of the barrier heights in each environment shows a clear correlation between TS stabilization and the barrier height. The average differential TS stabilization is 7.3 kcal mol(-1) in the enzyme. This is significantly higher than the small amount of TS stabilization in water (on average only 1.0 kcal mol(-1) relative to the substrate). The TS is stabilized mainly by electrostatic interactions with active site residues in the enzyme, with Arg90, Arg7 and Glu78 generally the most important. Conformational effects (e.g. strain of the substrate in the enzyme) do not contribute significantly to the lower barrier observed in the enzyme. The results show that catalysis is mainly due to better TS stabilization by the enzyme.