Phenolic Substrates Research Articles

Heterologous Expression and Characterization of a Laccase from Laccaria bicolor in Pichia pastoris and Arabidopsis thaliana.

Laccases can oxidize a variety of phenolic and non-phenolic substrates including synthetic dyes. In this research, a laccase gene Lcc9 from Laccaria bicolor was chemically synthesized and optimized to heterogeneous expression in Pichia pastoris and Arabidopsis thaliana. The properties of recombinant laccase expressed by P. pastoris were investigated. The laccase activity was optimal at 3.6 pH and 40°C. It exhibited Km and Vmax values of 0.565 mmol l⁻¹ and 1.51 μmol l⁻¹ min⁻¹ for ABTS respectively. As compared with untransformed control plants, the laccase activity in crude extracts of transgenic lines exhibited a 5.4 to 12.4-fold increase. Both laccases expressed in transgenic P. pastoris or A. thaliana could decolorize crystal violet. These results indicated that L. bicolor laccase gene may be transgenically exploited in fungi or plants for dye decolorization.

Drug and Chemical Glucosidation by Control Supersomes and Membranes from Spodoptera frugiperda (Sf) 9 Cells: Implications for the Apparent Glucuronidation of Xenobiotics by UDP-glucuronosyltransferase 1A5.

Accumulating evidence indicates that several human UDP-glucuronosyltransferase (UGT) enzymes catalyze both glucuronidation and glucosidation reactions. Baculovirus-infected insect cells [Trichoplusia ni and Spodoptera frugiperda (Sf9)] are used widely for the expression of recombinant human UGT enzymes. Following the observation that control Supersomes (c-SUP) express a native enzyme capable of glucosidating morphine, we characterized the glucosidation of a series of aglycones with a hydroxyl (aliphatic or phenolic), carboxylic acid, or amine functional group by c-SUP and membranes from uninfected Sf9 cells. Although both enzyme sources glucosidated the phenolic substrates investigated, albeit with differing activities, differences were observed in the selectivities of the native UDP-glucosyltransferases toward aliphatic alcohols, carboxylic acids, and amines. For example, zidovudine was solely glucosidated by c-SUP. By contrast, c-SUP lacked activity toward the amines lamotrigine and trifluoperazine and did not form the acyl glucoside of mycophenolic acid, reactions all catalyzed by uninfected Sf9 membranes. Glucosidation intrinsic clearances were high for several substrates, notably 1-hydroxypyrene (∼1400-1900 µl/min⋅mg). The results underscore the importance of including control cell membranes in the investigation of drug and chemical glucosidation by UGT enzymes expressed in T. ni (High-Five) and Sf9 cells. In a coincident study, we observed that UGT1A5 expressed in Sf9, human embryonic kidney 293T, and COS7 cells lacked glucuronidation activity toward prototypic phenolic substrates. However, Sf9 cells expressing UGT1A5 glucosidated 1-hydroxypyrene with UDP-glucuronic acid as the cofactor, presumably due to the presence of UDP-glucose as an impurity. Artifactual glucosidation may explain, at least in part, a previous report of phenolic glucuronidation by UGT1A5.

Structure and Catalytic Mechanism of a Bacterial Friedel-Crafts Acylase.

C−C bond‐forming reactions are key transformations for setting up the carbon frameworks of organic compounds. In this context, Friedel–Crafts acylation is commonly used for the synthesis of aryl ketones, which are common motifs in many fine chemicals and natural products. A bacterial multicomponent acyltransferase from Pseudomonas protegens (PpATase) catalyzes such Friedel–Crafts C‐acylation of phenolic substrates in aqueous solution, reaching up to >99 % conversion without the need for CoA‐activated reagents. We determined X‐ray crystal structures of the native and ligand‐bound complexes. This multimeric enzyme consists of three subunits: PhlA, PhlB, and PhlC, arranged in a Phl(A2C2)2B4 composition. The structure of a reaction intermediate obtained from crystals soaked with the natural substrate 1‐(2,4,6‐trihydroxyphenyl)ethanone together with site‐directed mutagenesis studies revealed that only residues from the PhlC subunits are involved in the acyl transfer reaction, with Cys88 very likely playing a significant role during catalysis. These structural and mechanistic insights form the basis of further enzyme engineering efforts directed towards enhancing the substrate scope of this enzyme.

A structural-chemical explanation of fungal laccase activity

Fungal laccases (EC 1.10.3.2) are multi-copper oxidases that oxidize a wide variety of substrates. Despite extensive studies, the molecular basis for their diverse activity is unclear. Notably, there is no current way to rationally predict the activity of a laccase toward a given substrate. Such knowledge would greatly facilitate the rational design of new laccases for technological purposes. We report a study of three datasets of experimental Km values and activities for Trametes versicolor and Cerrena unicolor laccase, using a range of protein modeling techniques. We identify diverse binding modes of the various substrates and confirm an important role of Asp-206 and His-458 (T. versicolor laccase numbering) in guiding substrate recognition. Importantly, we demonstrate that experimental Km values correlate with binding affinities computed by MMGBSA. This confirms the common assumption that the protein-substrate affinity is a major contributor to observed Km. From quantitative structure-activity relations (QSAR) we identify physicochemical properties that correlate with observed Km and activities. In particular, the ionization potential, shape, and binding affinity of the substrate largely determine the enzyme’s Km for the particular substrate. Our results suggest that Km is not just a binding constant but also contains features of the enzymatic activity. In addition, we identify QSAR models with only a few descriptors showing that phenolic substrates employ optimal hydrophobic packing to reach the T1 site, but then require additional electronic properties to engage in the subsequent electron transfer. Our results advance our ability to model laccase activity and lend promise to future rational optimization of laccases toward phenolic substrates.

Click. Coordinate. Catalyze. Using CuAAC Click Ligands in Small‐Molecule Model Chemistry of Tyrosinase

AbstractThree triazolylmethylpyridine ligands are synthesized using the copper‐catalyzed azide‐alkyne cycloaddition (CuAAC). The corresponding copper(I) complexes are investigated as catalysts for the oxygenation of several monophenols, in analogy to the enzyme tyrosinase. Importantly, they show a higher catalytic activity than previously investigated systems. This is ascribed to the lower charge donation of the electron‐poor triazole heterocycle, supporting the hydroxylation of phenolic substrates by an electrophilic substitution mechanism.

A comparative structural analysis of the surface properties of asco-laccases.

Laccases of different biological origins have been widely investigated and these studies have elucidated fundamentals of the generic catalytic mechanism. However, other features such as surface properties and residues located away from the catalytic centres may also have impact on enzyme function. Here we present the crystal structure of laccase from Myceliophthora thermophila (MtL) to a resolution of 1.62 Å together with a thorough structural comparison with other members of the CAZy family AA1_3 that comprises fungal laccases from ascomycetes. The recombinant protein produced in A. oryzae has a molecular mass of 75 kDa, a pI of 4.2 and carries 13.5 kDa N-linked glycans. In the crystal, MtL forms a dimer with the phenolic substrate binding pocket blocked, suggesting that the active form of the enzyme is monomeric. Overall, the MtL structure conforms with the canonical fold of fungal laccases as well as the features specific for the asco-laccases. However, the structural comparisons also reveal significant variations within this taxonomic subgroup. Notable differences in the T1-Cu active site topology and polar motifs imply molecular evolution to serve different functional roles. Very few surface residues are conserved and it is noticeable that they encompass residues that interact with the N-glycans and/or are located at domain interfaces. The N-glycosylation sites are surprisingly conserved among asco-laccases and in most cases the glycan displays extensive interactions with the protein. In particular, the glycans at Asn88 and Asn210 appear to have evolved as an integral part of the asco-laccase structure. An uneven distribution of the carbohydrates around the enzyme give unique properties to a distinct part of the surface of the asco-laccases which may have implication for laccase function–in particular towards large substrates.

Transformation of humic acids by two-domain laccase from Streptomyces anulatus

Abstract The properties of two-domain laccase of Streptomyces anulatus (SaSL) and its role in transformation of humic acids (HA) from chernozem, sod-podzolic soil and peat at alkaline pH values were studied. The SaSL was cloned, expressed in E. coli and obtained in an electrophoretically homogeneous state. SaSL is an oligomeric protein with a molecular weight of 235-300 kDa and six or eight subunits in the molecule. The molecular weight of the subunit is 37.7 kDa. Its catalytic properties are similar to those of the previously described two-domain laccase. The enzyme catalyzed oxidation of electron donors (K4[Fe(CN)6], ABTS) at acidic pH and phenolic substrates (2-methoxyphenol, 2,6-dimethixyphenol) at alkaline pH values. The efficiency of catalysis was higher in the case of electron donors than phenolic substrates. The enzyme showed a high thermal stability and was more stable at neutral and alkaline pH values. The enzyme effectively transformed humic acids at alkaline pH values. It was found that polymerization reactions took place during interaction of SaSL with HA, as well as with their high-molecular weight (>80 kDa) and low-molecular weight (

Inactivation of membrane‐bound and soluble polyphenol oxidases in apple (Malus domestica Borkh) by radio frequency processing for improved juice quality

AbstractRadio frequency (RF) processing was proposed to inactivate polyphenol oxidase (PPO) in apple tissue for improved juice quality. PPO in apple tissue exists as membrane‐bound PPO (mPPO) and soluble PPO (sPPO); mPPO is bound in chloroplast membranes and separated from phenolic substrates, preventing catalytic reaction, whereas sPPO is soluble in lumen. When cells break down, mPPO is released and comes in contact with phenolic substrates, contributing to browning and discoloration in juice. Research indicates that after RF treatment, inactivation of mPPO and sPPO in apple tissue was easier to achieve than extracted PPO. In addition, mPPO exhibited a lower thermal stability than that of sPPO. Treatment with RF for 10 min in apple tissue could effectively inactivate PPO and maintain a higher quality of squeezed apple juice than that of freshly squeezed juice treated by conventional thermal methods (65 °C for 10 min and 75 °C for 10 min).Practical applicationsIn fruit and vegetable processing industry, enzymatic browning is a difficult issue that leads to the deterioration of product quality. RF treatment can inactivate PPO rapidly in food and agricultural products to inhibit browning without heat conduction and overheated food surfaces, keeping high quality of the products. Results in this study could provide references for the application of RF technology on fruit and vegetable processing industry to inactivate endogenous enzyme.

Phomopsis longanae Chi-Induced Change in ROS Metabolism and Its Relation to Pericarp Browning and Disease Development of Harvested Longan Fruit.

Phomopsis longanae Chi is a major pathogenic fungus that infects harvested longan fruit. This study aimed to investigate the effects of P. longanae on reactive oxygen species (ROS) metabolism and its relation to the pericarp browning and disease development of harvested longan fruit during storage at 28°C and 90% relative humidity. Results showed that compared to the control longans, P. longanae-inoculated longans displayed higher indexes of pericarp browning and fruit disease, higher O2-. generation rate, higher accumulation of malondialdehyde (MDA), lower contents of glutathione (GSH) and ascorbic acid (AsA), lower 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical scavenging ability and reducing power in pericarp. In addition, P. longanae-infected longans exhibited higher activities of superoxide dismutase (SOD), catalase (CAT), and ascorbate peroxidase (APX) in the first 2 days of storage, and lower activities of SOD, CAT, and APX during storage day 2–5 than those in the control longans. These findings indicated that pericarp browning and disease development of P. longanae-infected longan fruit might be the result of the reducing ROS scavenging ability and the increasing O2-. generation rate, which might lead to the peroxidation of membrane lipid, the loss of compartmentalization in longan pericarp cells, and subsequently cause polyphenol oxidase (PPO) and peroxidase (POD) to contact with phenolic substrates which result in enzymatic browning of longan pericarp, as well as cause the decrease of disease resistance to P. longanae and stimulate disease development of harvested longan fruit.

Unprecedented direct cupric-superoxo conversion to a bis-μ-oxo dicopper(III) complex and resulting oxidative activity

Investigations of small molecule copper-dioxygen chemistry can and have provided fundamental insights into enzymatic processes (e.g., copper metalloenzyme dioxygen binding geometries and their associated spectroscopy and substrate reactivity). Strategically designing copper-binding ligands has allowed for insight into properties that favor specific (di)copper-dioxygen species. Herein, the tetradentate tripodal TMPA-based ligand (TMPA = tris((2-pyridyl)methyl)amine) possessing a methoxy moiety in the 6-pyridyl position on one arm (OCH3TMPA) was investigated. This system allows for a trigonal bipyramidal copper(II) geometry as shown by the UV–vis and EPR spectra of the cupric complex [(OCH3TMPA)CuII(OH2)](ClO4)2. Cyclic voltammetry experiments determined the reduction potential of this copper(II) species to be −0.35 V vs. Fc+/0 in acetonitrile, similar to other TMPA-derivatives bearing sterically bulky 6-pyridyl substituents. The copper-dioxygen reactivity is also analogous to these TMPA-derivatives, affording a bis-μ-oxo dicopper(III) complex, [{(OCH3TMPA)CuIII}2(O2−)2]2+, upon oxygenation of the copper(I) complex [(OCH3TMPA)CuI](B(C6F5)4) at cryogenic temperatures in 2-methyltetrahydrofuran. This highly reactive intermediate is capable of oxidizing phenolic substrates through a net hydrogen atom abstraction. However, after bubbling of the precursor copper(I) complex with dioxygen at very low temperatures (−135 °C), a cupric superoxide species, [(OCH3TMPA)CuII(O2−)]+, is initially formed before slowly converting to [{(OCH3TMPA)CuIII}2(O2−)2]2+. This appears to be the first instance of the direct conversion of a cupric superoxide to a bis-μ-oxo dicopper(III) species in copper(I)-dioxygen chemistry.

Rapid characterization of the activities of lignin-modifying enzymes based on nanostructure-initiator mass spectrometry (NIMS)

BackgroundProducing valuable fuels and chemicals from lignin is a key factor for making lignocellulosic biomass economically feasible; however, significant roadblocks exist due to our lack of detailed understanding of how lignin is enzymatically depolymerized and of the range of possible lignin fragments that can be produced. Development of suitable enzymatic assays for characterization of putative lignin active enzymes is an important step towards improving our understanding of the catalytic activities of relevant enzymes. Previously, we have successfully built an assay platform based on glycan substrates containing a charged perfluorinated tag and nanostructure-initiator mass spectrometry to study carbohydrate active enzymes, especially various glycosyl hydrolyses. Here, we extend this approach to develop a reliable and rapid assay to study lignin-modifying enzymes.ResultsTwo β-aryl ether bond containing model lignin dimer substrates, designed to be suitable for studying the activities of lignin-modifying enzymes (LMEs) by nanostructure-initiator mass spectrometry (NIMS), were successful synthesized. Small-angle neutron scattering experiments showed that these substrates form micelles in solution. Two LMEs, laccase from the polypore mushroom Trametes versicolor, and manganese peroxidase (MnP) from white rot fungus Nematoloma frowardii, were tested for catalytic activity against the two model substrates. We show that the reaction of laccase and MnP with phenolic substrate yields products that arise from the cleavage of the carbon–carbon single bond between the α-carbon and the adjacent aryl carbon, consistent with the mechanism for producing phenoxy radical as reaction intermediates. Reactions of the nonphenolic substrate with laccase, on the other hand, adopt a different pathway by producing an α-oxidation product; as well as the cleavage of the β-aryl ether bond. No cleavage of the carbon–carbon bond between the α-carbon and the aryl carbon was observed. To facilitate understanding of reaction kinetics, the reaction time course for laccase activity on the phenolic substrate (I) was generated by the simultaneous measurement of all products at different time points of the reaction. Withdrawal of only a small sample aliquot (0.2 μL at each time point) ensured minimum perturbation of the reaction. The time course can help us to understand the enzyme kinetics.ConclusionsA new assay procedure has been developed for studying lignin-modifying enzymes by nanostructure-initiator mass spectrometry. Enzyme assays of a laccase and a MnP on phenolic and nonphenolic β-aryl ether substrates revealed different primary reaction pathways due to the availability of the phenoxy radical intermediates. Our assay provides a wealth of information on bond cleavage events not available using conventional colorimetric assays and can easily be carried out in microliter volumes and the quantitative analysis of product formation and kinetics is rapidly achieved by NIMS. This is the first time that NIMS technology was applied to study the activities of lignin-modifying enzymes. Unlike other previous works, our use of amphiphilic guaiacylglycerol β-O-4 substrate (I) enables the formation of micelles. This approach helps avoid the re-polymerization of the resulting monomeric product. As a result, our assay can clearly demonstrate the degradation pathways of phenolic guaiacylglycerol β-O-4 type of molecules with laccase and MnP.

Effect of Dipping Pre-treatment with Unripe Grape Juice on Dried “Golden Delicious” Apple Slices

The consumption of apple chips is increasing in the last decades because of the consumer’s preferences towards healthy snacks. However, the enzymatic browning, which is attributed mainly to polyphenol oxidase (PPO, EC 1.10.3.1) activity, negatively affects the sensory, nutritional, and antioxidant properties of dried apples as a consequence of the oxidation of phenolic substrates during post-harvest stages. The antibrowning and antioxidant effects of dipping pre-treatments with unripe grape juice as well as reference formulations including a mixture of 1% w/v ascorbic acid and 1% w/v citric acid, 6% w/v NatureSeal®, and 0.1% w/v sodium metabisulfite were evaluated by spectrophotometric and colorimetric assays on dried “Golden Delicious” apple slices. Based on HPLC analysis, the treated apple slices were enriched with bioactive compounds which effectively improved antioxidant capacity and limited enzymatic browning by increasing lightness, reducing color change, and inhibiting PPO activity. Hence, the juice of unripe grapes recovered as agricultural wastes during bunch thinning of vineyards could be proposed as a safe, inexpensive, and eco-friendly alternative to conventional antibrowning treatments in apple processing.

Biochemical characterization of Burkina red radish (<i>Raphanus sativus</i>) peroxidase

Objectives: Peroxidases (POX), isoenzymes were purified to homogeneity from bulbs of Raphanus sativus (Radish) grown in Burkina Faso and characterized for use as an alternative source of POX for biotechnological applications. Methodology and Results: The enzymes were purified using cation exchange, anion-exchange and hydrophobic interaction chromatography. Two isoperoxidases, cationic POX1 and anionic POX2, were isolated and purified 18.5 and 27.2 fold, respectively. Purified enzymes were found to be monomeric proteins with molecular masses of 70 kDa and 47 kDa for POX1 and POX2, respectively, as determined by SDS-PAGE. The effect of pH and temperature was done using guaicol as a substrate. The optimal pH of the both purified POX was 5.6 and 80 % of its activity was retained at pH values between 4.0–8.0 after incubation for 2 h, but POX2 appeared to be highly stable than POX1. POX1 and POX2 had optimum temperatures of activity at 35° and 40°C, respectively. Activities of these isoenzymes were determined using different phenolic substrates in the presence and absence of ionic effectors. The results show that both POX activities were activated by bivalent cations such as Mg 2+ and Ca 2+ but were inhibited by K + , Na + , Zn 2+ , EDTA and reducing compounds. While Ba 2+ is an activator for POX2, it had no effect on POX1. They oxidize all the phenolic compounds used. The greatest rate was obtained with ABTS and guaiacol, for POX1 and POX2, respectively. Conclusions and application of findings: POXs oxidize a wide range of phenolic substrates and have high stability at a wide range of pH. These properties make these enzymes potential biotechnological tools. They could be use in industrial applications to produce food dyes, phenolic resins from natural phenolic compounds or in the construction of biosensors and kits for analyses and diagnostics. They could play also an important role in the oxidation of phenolic compounds in polluted water. This is interesting because it could be useful in finding solutions to the thorny problem of recalcitrant phenolic compounds that resist to conventional methods for bioremediation. Keywords: peroxidase, purification, characterization, Raphanus sativus , phenolic compound

Rhodium(III)-Catalyzed Meta-Selective C-H Alkenylation of Phenol Derivatives.

Rhodium(III)-catalyzed remote meta-selective-C-H alkenylation of phenol derivatives has been developed using a traceless organosilicon template as the directing group. This transformation proceeds smoothly with good yields and high meta-selectivities toward a series of phenol and alkene substrates. In addition, this protocol provides an effective strategy for late-stage transformations of various meta-alkenylated aromatic compounds.

Bacterial laccases: promising biological green tools for industrial applications.

Multicopper oxidases (MCOs) are a pervasive family of enzymes that oxidize a wide range of phenolic and nonphenolic aromatic substrates, concomitantly with the reduction of dioxygen to water. MCOs are usually divided into two functional classes: metalloxidases and laccases. Given their broad substrate specificity and eco-friendliness (molecular oxygen from air as is used as the final electron acceptor and they only release water as byproduct), laccases are regarded as promising biological green tools for an array of applications. Among these laccases, those of bacterial origin have attracted research attention because of their notable advantages, including broad substrate spectrum, wide pH range, high thermostability, and tolerance to alkaline environments. This review aims to summarize the significant research efforts on the properties, mechanisms and structures, laccase-mediator systems, genetic engineering, immobilization, and biotechnological applications of the bacteria-source laccases and laccase-like enzymes, which principally include Bacillus laccases, actinomycetic laccases and some other species of bacterial laccases. In addition, these enzymes may offer tremendous potential for environmental and industrial applications.

Moderate vacuum packing and low temperature effects on qualities of harvested mung bean (Vigna radiata L.) sprouts

Post-harvest browning, decay, lignification, and other adverse physiological effects limit the shelf-life of mung bean sprouts at ambient temperature. In this study, vacuum packing at pressures of 0, ↓0.02, ↓0.04, and ↓0.06 mPa at 20 °C, and the optimum pressure of ↓0.04 mPa at 4 °C for maintenance of sprouts, was investigated. Chlorogenic acid was identified as the main phenolic compound leading to the browning of mung bean sprouts, along with total flavonoids, total phenols, rutin, and o-phthalic acid. Among these, flavonoids were identified as being the main substrate for polyphenol oxidase (PPO) activity. Moderate vacuum packing combined with low temperature storage inhibited PPO, as well as peroxidase (POD) and superoxide dismutase activities, which was associated with a slower rate of browning. In addition, these storage conditions reduced the activity of phenylalanine ammonia lyase (PAL), thereby suppressing the synthesis of phenolic substrates used in the browning reaction. Moderate vacuum packing combined with low temperature storage also maintained the activities of the cell wall-degrading enzymes cellulase and xylanase, and suppressed POD and PAL activities. Together, these effects would be expected to lead to a decrease in cellulose, hemicellulose, and lignin levels, ultimately restricting sprout elongation and maintaining the firmness of the sprouts.

Purification and biochemical characterization of peroxidase isoenzymes from Ficus carica latex

Abstract Three peroxidase isoenzymes were isolated from Ficus carica latex using CM-Sepharose, DEAE-Sepharose and Sephacryl S-200. The complete purification was carried out for FP1 only due to the low level of activity and protein concentration of FP2 and FP3. The purified isoenzyme FP1 was found to be monomeric with a molecular weight of 30 kDa. FP1 and FP3 isoenzymes had the same pH and temperature optima at pH 5.5 and 40 °C, whereas the optimum values of pH and temperature were at pH 7.0 and 30 °C for FP2. On the other hand, FP1, FP2 and FP3 were stable at 50 °C, 40 °C and 30 °C respectively, whereas FP3 had low thermostability. FP1 isoenzyme was found to be stable between pH 5.0 and 7.5, and FP2 was stable from pH 4.0–8.0, while FP3 was found to be stable in acidic range between pH 4.5 and pH 5.5. The activity of both FP1 and FP2 peroxidase isoenzymes was increased by the high concentration of Ca2+ (10 mM). The three peroxidase isoenzymes have a broad specificity towards some phenolic substrates and O -Phenylenediamine showed higher affinity towards the three peroxidase isoenzymes, with Km values of 3.87, 3.64 and 3.33 mM for FP1, FP2 and FP3. The properties of these isoenzymes meet the prerequisites needed for environmental and industrial applications.

Peroxidase versus Peroxygenase Activity: Substrate Substituent Effects as Modulators of Enzyme Function in the Multifunctional Catalytic Globin Dehaloperoxidase.

The dehaloperoxidase-hemoglobin (DHP) from the terebellid polychaete Amphitrite ornata is a multifunctional hemoprotein that catalyzes the oxidation of a wide variety of substrates, including halo/nitrophenols, haloindoles, and pyrroles, via peroxidase and/or peroxygenase mechanisms. To probe whether substrate substituent effects can modulate enzyme activity in DHP, we investigated its reactiviy against a panel of o-guaiacol substrates given their presence (from native/halogenated and non-native/anthropogenic sources) in the benthic environment that A. ornata inhabits. Using biochemical assays supported by spectroscopic, spectrometric, and structural studies, DHP was found to catalyze the H2O2-dependent oxidative dehalogenation of 4-haloguaiacols (F, Cl, and Br) to 2-methoxybenzoquinone (2-MeOBQ). 18O labeling studies confirmed that O atom incorporation was derived exclusively from water, consistent with substrate oxidation via a peroxidase-based mechanism. The 2-MeOBQ product further reduced DHP to its oxyferrous state, providing a link between the substrate oxidation and O2 carrier functions of DHP. Nonnative substrates resulted in polymerization of the initial substrate with varying degrees of oxidation, with 2-MeOBQ identified as a minor product. When viewed alongside the reactivity of previously studied phenolic substrates, the results presented here show that simple substituent effects can serve as functional switches between peroxidase and peroxygenase activities in this multifunctional catalytic globin. More broadly, when recent findings on DHP activity with nitrophenols and azoles are included, the results presented here further demonstrate the breadth of heterocyclic compounds of anthropogenic origin that can potentially disrupt marine hemoglobins or function as environmental stressors, findings that may be important when assessing the environmental impact of these pollutants (and their metabolites) on aquatic systems.

Laccase-catalyzed bioconjugation of tyrosine-tagged functional proteins

The site-specific cross-linking of functional proteins creates macromolecular assemblies that exhibit unique biochemical and/or physicochemical properties. Herein, we explored the potential of laccase as a biocatalyst for the site-specific cross-linking of tyrosine-tagged proteins. Trametes sp. laccase (TL) was selected as the cross-linking catalyst, and Escherichia coli alkaline phosphatase (BAP) and antibody-binding proteins (pG2pAs) were employed as model proteins. The protein models were genetically fused to a peptide tag containing a tyrosine residue (Y-tag) at the N- and/or C-termini. Proteins without Y-tags were used as controls. The Y-tagged proteins could be recognized by TL as macromolecular substrates, leading to the oxidative formation of protein polymers, whereas no polymerization was observed with intact BAP or pG2pA. The TL-catalyzed cross-linking of Y-tagged proteins proceeded at a relatively high pH in comparison with that of small phenolic substrates. Co-polymers of BAP and pG2pA were able to be prepared by mixing the aqueous solution of each component in the presence of TL. A combination of bis-Y-tagged pG2pA (Y-pG2pA-Y) and Y-tagged BAP (BAP-Y) yielded functional co-polymers compatible with enzyme-linked immunosorbent assay (ELISA). The detection limit of the ELISA of ovalbumin with anti-OVA IgG depended on the molar ratio of BAP-Y and Y-pG2pA-Y in the TL-catalyzed cross-linking reaction. A high molar ratio of BAP-Y to Y-pG2pA-Y (75:1) resulted in the highest absorbance in the ELISA. The results suggested that the formation of a bifunctional protein polymer with a high molar ratio of signaling unit to antibody-binding unit gave better performance in antigen detection than using lower ratios.

Multiple Reaction Monitoring for quantitative laccase kinetics by LC-MS

Laccases (EC 1.10.3.2) are enzymes known for their ability to catalyse the oxidation of phenolic compounds using molecular oxygen as the final electron acceptor. Lignin is a natural phenylpropanoids biopolymer whose degradation in nature is thought to be aided by enzymatic oxidation by laccases. Laccase activity is often measured spectrophotometrically on compounds such as syringaldazine and ABTS which poorly relate to lignin. We employed natural phenolic hydroxycinnamates having different degree of methoxylations, p-coumaric, ferulic and sinapic acid, and a lignin model OH-dilignol compound as substrates to assess enzyme kinetics by HPLC-MS on two fungal laccases Trametes versicolor laccase, Tv and Ganoderma lucidum laccase, Gl. The method allowed accurate kinetic measurements and detailed insight into the product profiles of both laccases. Both Tv and Gl laccase are active on the hydroxycinnammates and show a preference for substrate with methoxylations. Product profiles were dominated by the presence of dimeric and trimeric species already after 10 minutes of reaction and similar profiles were obtained with the two laccases. This new HPLC-MS method is highly suitable and accurate as a new method for assaying laccase activity on genuine phenolic substrates, as well as a tool for examining laccase oxidation product profiles.