Integrated Continuous-Flow Production of Wax Esters Combining Whole-Cell and <i>In Vitro</i> Biocatalysis

A first-of-its-kind, fully continuous synthesis of wax esters from biobased precursors (glucose, fatty acids) was developed using metabolically engineered cells and in vitro enzyme catalysis. The cells, overexpressing fatty acyl-CoA reductase and xylose reductase, could be immobilized onto polyesters and packed in a continuous reactor. The immobilized cells were employed in the bioconversion, incorporating in situ extraction using dodecane as the solvent. Such extractive bioconversion was capable of producing fatty alcohols continuously at a productivity of 8.2 mg/(L·h). The immiscible aqueous-dodecane flow stream from the extractive bioconversion was then separated using an in-line membrane-based separator. The dodecane-rich phase was directed into an enzymatic reactor containing Novozyme 435 for the esterification of fatty alchols and fatty acids into the wax esters. A continuous production of wax esters (6.38–23.35 mg/(L·h)) was achieved as a result of the successful streamlining of the cascade biocatalytic process

Mechanism of Oxygen Activation in a Flavin-Dependent Monooxygenase: A Nearly Barrierless Formation of C4a-Hydroperoxyflavin via Proton-Coupled Electron Transfer

Understanding how flavin-dependent enzymes activate oxygen for their oxidation and oxygenation reactions is one of the most challenging issues in flavoenzymology. Density functional calculations and transient kinetics were performed to investigate the mechanism of oxygen activation in the oxygenase component (C₂) of <i>p</i>-hydroxyphenylacetate 3-hydroxylase (HPAH). We found that the protonation of dioxygen by His396 via a proton-coupled electron transfer mechanism is the key step in the formation of the triplet diradical complex of flavin semiquinone and ^•OOH. This complex undergoes intersystem crossing to form the open-shell singlet diradical complex before it forms the closed-shell singlet C4a-hydroperoxyflavin intermediate (C4aOOH). Notably, density functional calculations indicated that the formation of C4aOOH is nearly barrierless, possibly facilitated by the active site arrangement in which His396 positions the proximal oxygen of the ^•OOH in an optimum position to directly attack the C4a atom of the isoalloxazine ring. The nearly barrierless formation of C4aOOH agrees well with the experimental results; based on transient kinetics and Eyring plot analyses, the enthalpy of activation for the formation of C4aOOH is only 1.4 kcal/mol and the formation of C4aOOH by C₂ is fast (∼10⁶ M^–1 s^–1 at 4 °C). The calculations identified Ser171 as the key residue that stabilizes C4aOOH by accepting a hydrogen bond from the H­(N5) of the isoalloxazine ring. Both Ser171 and Trp112 facilitate H₂O₂ elimination by donating hydrogen bonds to the proximal oxygen of the OOH moiety during the proton transfer. According to our combined theoretical and experimental studies, the existence of a positively charged general acid at the position optimized for facilitating the proton-coupled electron transfer has emerged as an important catalytic feature for the oxygen activation process in flavin-dependent enzymes

Control of C4a-Hydroperoxyflavin Protonation in the Oxygenase Component of <i>p</i>‑Hydroxyphenylacetate-3-hydroxylase

The protonation status of the peroxide moiety in C4a-(hydro)­peroxyflavin of <i>p</i>-hydroxyphenylacetate-3-hydroxylase can be directly monitored using transient kinetics. The p<i>K</i>_a for the wild-type (WT) enzyme is 9.8 ± 0.2, while the values for the H396N, H396V, and H396A variants are 9.3 ± 0.1, 7.3 ± 0.2, and 7.1 ± 0.2, respectively. The hydroxylation efficiency of these mutants is lower than that of the WT enzyme. Solvent kinetic isotope effect studies indicate that proton transfer is not the rate-limiting step in the formation of C4a-OOH. All data suggest that His396 may act as an instantaneous proton provider for the proton-coupled electron transfer that occurs before the transition state of C4a-OOH formation

A Single-Site Mutation at Ser146 Expands the Reactivity of the Oxygenase Component of <i>p</i>‑Hydroxyphenylacetate 3‑Hydroxylase

The oxygenase component (C₂) of <i>p</i>-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from <i>Acinetobacter baumannii</i> catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C₂ with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (<i>k</i>_OH) for 4-APA of 0.028 s^–1 compared to 17 s^–1 for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s^–1 for 4-HPA versus 2.5 s^–1 for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C₂ reactivity toward a phenolic compound. Removing this hydroxyl group expands C₂ activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue

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

The Transfer of Reduced Flavin Mononucleotide from LuxG Oxidoreductase to Luciferase Occurs via Free Diffusion

Bacterial luciferase (LuxAB) is a two-component flavin mononucleotide (FMN)-dependent monooxygenase that catalyzes the oxidation of reduced FMN (FMNH^–) and a long-chain aliphatic aldehyde by molecular oxygen to generate oxidized FMN, the corresponding aliphatic carboxylic acid, and concomitant emission of light. The LuxAB reaction requires a flavin reductase to generate FMNH^– to serve as a luciferin in its reaction. However, FMNH^– is unstable and can react with oxygen to generate H₂O₂, so that it is important to transfer it efficiently to LuxAB. Recently, LuxG has been identified as a NADH:FMN oxidoreductase that supplies FMNH^– to luciferase <i>in vivo</i>. In this report, the mode of transfer of FMNH^– between LuxG from <i>Photobacterium leiognathi</i> TH1 and LuxABs from both <i>P. leiognathi</i> TH1 and <i>Vibrio campbellii</i> (<i>Pl</i>LuxAB and <i>Vc</i>LuxAB, respectively) was investigated using single-mixing and double-mixing stopped-flow spectrophotometry. The oxygenase component of <i>p</i>-hydroxyphenylacetate hydroxylase (C2) from <i>Acinetobacter baumannii</i>, which has no structural similarity to LuxAB, was used to measure the kinetics of release of FMNH^– from LuxG. With all FMNH^– acceptors used (C₂, <i>Pl</i>LuxAB, and <i>Vc</i>LuxAB), the kinetics of FMN reduction on LuxG were the same, showing that LuxG releases FMNH^– with a rate constant of 4.5–6 s^–1. Our data showed that the kinetics of binding of FMNH^–to <i>Pl</i>LuxAB and <i>Vc</i>LuxAB and the subsequent reactions with oxygen were the same with either free FMNH^– or FMNH^– generated <i>in situ</i> by LuxG. These results strongly suggest that no complexes between LuxG and the various species are necessary to transfer FMNH^– to the acceptors. The kinetics of the overall reactions and the individual rate constants correlate well with a free diffusion model for the transfer of FMNH^– from LuxG to either LuxAB

Tuned Amperometric Detection of Reduced β‑Nicotinamide Adenine Dinucleotide by Allosteric Modulation of the Reductase Component of the <i>p</i>‑Hydroxyphenylacetate Hydroxylase Immobilized within a Redox Polymer

We report the fabrication of an amperometric NADH biosensor system that employs an allosterically modulated bacterial reductase in an adapted osmium­(III)-complex-modified redox polymer film for analyte quantification. Chains of complexed Os­(III) centers along matrix polymer strings make electrical connection between the immobilized redox protein and a graphite electrode disc, transducing enzymatic oxidation of NADH into a biosensor current. Sustainable anodic signaling required (1) a redox polymer with a formal potential that matched the redox switch of the embedded reductase and avoided interfering redox interactions and (2) formation of a cross-linked enzyme/polymer film for stable biocatalyst entrapment. The activity of the chosen reductase is enhanced upon binding of an effector, i.e. <i>p</i>-hydroxy-phenylacetic acid (<i>p</i>-HPA), allowing the acceleration of the substrate conversion rate on the sensor surface by in situ addition or preincubation with <i>p</i>-HPA. Acceleration of NADH oxidation amplified the response of the biosensor, with a 1.5-fold increase in the sensitivity of analyte detection, compared to operation without the allosteric modulator. Repetitive quantitative testing of solutions of known NADH concentration verified the performance in terms of reliability and analyte recovery. We herewith established the use of allosteric enzyme modulation and redox polymer-based enzyme electrode wiring for substrate biosensing, a concept that may be applicable to other allosteric enzymes

3,4-Dihydroxyphenylacetate 2,3-dioxygenase from <i>Pseudomonas aeruginosa</i>: An Fe(II)-containing enzyme with fast turnover

<div><p>3,4-dihydroxyphenylacetate (DHPA) dioxygenase (DHPAO) from <i>Pseudomonas aeruginosa</i> (PaDHPAO) was overexpressed in <i>Escherichia coli</i> and purified to homogeneity. As the enzyme lost activity over time, a protocol to reactivate and conserve PaDHPAO activity has been developed. Addition of Fe(II), DTT and ascorbic acid or ROS scavenging enzymes (catalase or superoxide dismutase) was required to preserve enzyme stability. Metal content and activity analyses indicated that PaDHPAO uses Fe(II) as a metal cofactor. NMR analysis of the reaction product indicated that PaDHPAO catalyzes the 2,3-extradiol ring-cleavage of DHPA to form 5-carboxymethyl-2-hydroxymuconate semialdehyde (CHMS) which has a molar absorptivity of 32.23 mM^-1cm^-1 at 380 nm and pH 7.5. Steady-state kinetics under air-saturated conditions at 25°C and pH 7.5 showed a <i>K</i>_m for DHPA of 58 ± 8 μM and a <i>k</i>_cat of 64 s^-1, indicating that the turnover of PaDHPAO is relatively fast compared to other DHPAOs. The pH-rate profile of the PaDHPAO reaction shows a bell-shaped plot that exhibits a maximum activity at pH 7.5 with two p<i>K</i>_a values of 6.5 ± 0.1 and 8.9 ± 0.1. Study of the effect of temperature on PaDHPAO activity indicated that the enzyme activity increases as temperature increases up to 55°C. The Arrhenius plot of ln(<i>k’</i>_cat) <i>versu</i>s the reciprocal of the absolute temperature shows two correlations with a transition temperature at 35°C. Two activation energy values (<i>E</i>_a) above and below the transition temperature were calculated as 42 and 14 kJ/mol, respectively. The data imply that the rate determining steps of the PaDHPAO reaction at temperatures above and below 35°C may be different. Sequence similarity network analysis indicated that PaDHPAO belongs to the enzyme clusters that are largely unexplored. As PaDHPAO has a high turnover number compared to most of the enzymes previously reported, understanding its biochemical and biophysical properties should be useful for future applications in biotechnology.</p></div

Summary of PaDHPAO purification.

<p>Summary of PaDHPAO purification.</p

Effects of temperature and pH on the catalysis of PaDHPAO.

<p>(A) To evaluate the effects of temperature on activity, the <i>k’</i>_cat values were measured at 5–70°C and plotted as a function of temperature. The inset in A is the Arrhenius plot of ln(<i>k’</i>_cat) versus the reciprocal of the absolute temperature (5–55°C). The data indicate a transition temperature at 35°C (arrow). (B) To evaluate the effect of pH on activity, the apparent maximum velocities (<i>V’</i>_max) of the PaDHPAO reaction were measured under various pH conditions (4.0–10.5). A plot of the pH-rate profile indicates an optimal pH at 7.5 with two p<i>K</i>_a values of 6.5 ± 0.1 and 8.9 ± 0.1, respectively.</p