Bacterial Multicopper Oxidase Research Articles

Biogenic Manganese Oxide Synthesized by a Marine Bacterial Multicopper Oxidase MnxG Reveals Oxygen Evolution Activity

Biogenic Manganese Oxide Synthesized by a Marine Bacterial Multicopper Oxidase MnxG Reveals Oxygen Evolution Activity

Resolving metal binding properties within subunits of a multimeric enzyme Mnx by surface induced dissociation and native ion mobility mass spectrometry

Multi-subunit enzymes function as coordinated assemblies. Yet most enzymatic assays measure the summed output of all populations in solution and cannot easily differentiate contributions of individual subunits. Native mass spectrometry detects intact protein complexes in the gas phase. Surface induced dissociation further releases subunits from protein complexes while retaining compact conformations and bound ligands. Combined with ion mobility, the released subunits can then be carefully monitored for more in-depth structural analysis. Mnx is a unique bacterial multicopper oxidase complex that oxidizes Mn(II) to form MnO2 minerals, and is composed of three subunits: MnxG, a multicopper oxidase containing the active site, and two accessory proteins, MnxE and MnxF which also bind copper ions. Other known multicopper oxidases do not require accessory proteins, therefore the functions of MnxE and MnxF are not well understood. Here, we use native mass spectrometry with surface induced dissociation and ion mobility to characterize the metal binding properties of Mnx with two metals, catalytic Cu(II) and Mn(II) substrate. We demonstrate our assay can detect subtle structural changes within each subunit, which are presumably related to the allosteric mechanism. We also noticed that ionic strength and solution composition can impact metal binding and must be carefully investigated for such experiments.

The Genus Iodidimonas: From Its Discovery to Potential Applications.

The genus Iodidimonas was recently proposed in the class Alphaproteobacteria. Iodidimonas strains are aerobic, mesophilic, neutrophilic, moderately halophilic, and chemo-organotrophic. They were first discovered in natural gas brine water containing a very high level of iodide (I−). They exhibited a unique phenotypic feature of iodide oxidation to form molecular iodine (I2). Iodidimonas was also enriched and isolated from surface seawater supplemented with iodide, and it is clearer now that their common habitats are those enriched with iodide. In such environments, Iodidimonas species seem to attack microbial competitors with the toxic form I2 to occupy their ecological niche. The iodide-oxidizing enzyme (IOX) purified from the Iodidimonas sp. strain Q-1 exhibited high catalytic efficiency for iodide and consisted of at least two proteins IoxA and IoxC. IoxA is a putative multicopper oxidase with four conserved copper-binding regions but is phylogenetically distinct from other bacterial multicopper oxidases. The IOX/iodide system could be used as a novel enzyme-based antimicrobial system which can efficiently kill Bacillus spores. Furthermore, the IOX/iodide system can be applied to the decolorization of recalcitrant dyes, where iodide may function as a novel inorganic natural redox mediator.

Toward Characterizing the Genetic Basis of Trace Organic Contaminant Biotransformation in Activated Sludge: The Role of Multicopper Oxidases as a Case Study.

Activated sludge treatment leverages the ability of microbes to uptake and (co)-metabolize chemicals and has shown promise in eliminating trace organic contaminants (TrOCs) during wastewater treatment. However, targeted interventions to optimize the process are limited as the fundamental drivers of the observed reactions remain elusive. In this work, we present a comprehensive workflow for the identification and characterization of key enzymes involved in TrOCs biotransformation pathways in complex microbial communities. To demonstrate the applicability of the workflow, we investigated the role of the enzymatic group of multicopper oxidases (MCOs) as one putatively relevant driver of TrOCs biotransformation. To this end, we analyzed activated sludge metatranscriptomic data and selected, synthesized, and heterologously expressed three phylogenetically distinct MCO-encoding genes expressed in communities with different TrOCs oxidation potentials. Following the purification of the encoded enzymes, we screened their activities against different substrates. We saw that MCOs exhibit significant activities against selected TrOCs in the presence of the mediator compound 2,2'-azino-bis-3-ethylbenzthiazoline-6-sulfonic acid and, in some cases, also in the presence of the wastewater contaminant 4'-hydroxy-benzotriazole. In the first case, we identified oxidation products previously reported from activated sludge communities and concluded that in the presence of appropriate mediators, bacterial MCOs could contribute to the biological removal of TrOCs. Similar investigations of other key enzyme systems may significantly advance our understanding of TrOCs biodegradation and assist the rational design of biology-based water treatment strategies in the future.

Lignin-oxidizing activity of bacterial laccases characterized using soluble substrates and polymeric lignin

Efficient biotransformation of lignin requires the activity of different oxidative enzymes. In this work, 19 bacterial multi-copper oxidases were screened for oxidase activity against 19 soluble substrates and revealed the highest activity in the laccase CotABsu (BSU0630) from Bacillus subtilis. Structure-based site-directed mutagenesis of CotABsu identified four conserved residues (His419, Cys492, His497, and Met502) as critical for activity against 2,2′-azinobis(3-ethylbenzthiazoline-6-sulfonate) (ABTS). Greatly reduced oxidase activity was found in the CotABsu mutant proteins E213A, N214A, C229A, N264A, E298A, T415A, R416A, Q468A, and T480A. We also designed a lignin-agarose plate screen for detecting oxidase activity of purified proteins against polymeric lignin, which confirmed the results obtained with ABTS and identified three mutant variants with increased activity toward kraft lignin (E213A, T415A, and T260A). X-ray photoelectron spectroscopy analysis of low sulfonate kraft lignin after incubation with CotABsu revealed a reduction in the content of CC/CC bonds and increase in CO/CO bonds. Product analyses using mass spectrometry, liquid chromatography, and bright-field microscopy revealed an increased polymerization state of reaction products suggesting that formation of radical intermediates was followed by radical coupling. Our results provide further insights into the mechanisms of lignin oxidation by laccases.

An acid-stable bacterial laccase identified from the endophyte Pantoea ananatis Sd-1 genome exhibiting lignin degradation and dye decolorization abilities.

Isolation and identification of a novel laccase (namely Lac4) with various industrial applications potentials from an endophytical bacterium. Endophyte Sd-1 cultured in rice straw showed intra- and extra-cellular laccase activities. Genomic analysis of Sd-1 identified four putative laccases, Lac1 to Lac4. However, only Lac4 contains the complete signature sequence of laccase and shares at most 64% sequence identity with other characterized bacterial multi-copper oxidases. Recombinant Lac4 can oxidize non-phenolic and phenolic compounds under acidic conditions and at 30-50°C; Km values of Lac4 for ABTS at pH 2.5 and for guaiacol at pH 4.5 were 1±0.15 and 6.1±1.7mM, respectively. The activity of Lac4 was stimulated by 0.8mM Cu(2+) and 5mM Fe(2+). In addition, Lac4 could decolorize various synthetic dyes and exhibit the degradation rate of 38% for lignin. The data suggest that Lac4 possesses promising biotechnological potentials.

Surface Mn(II) oxidation actuated by a multicopper oxidase in a soil bacterium leads to the formation of manganese oxide minerals.

In this manuscript, we report that a bacterial multicopper oxidase (MCO266) catalyzes Mn(II) oxidation on the cell surface, resulting in the surface deposition of Mn(III) and Mn(IV) oxides and the gradual formation of bulky oxide aggregates. These aggregates serve as nucleation centers for the formation of Mn oxide micronodules and Mn-rich sediments. A soil-borne Escherichia coli with high Mn(II)-oxidizing activity formed Mn(III)/Mn(IV) oxide deposit layers and aggregates under laboratory culture conditions. We engineered MCO266 onto the cell surfaces of both an activity-negative recipient and wild-type strains. The results confirmed that MCO266 governs Mn(II) oxidation and initiates the formation of deposits and aggregates. By contrast, a cell-free substrate, heat-killed strains, and intracellularly expressed or purified MCO266 failed to catalyze Mn(II) oxidation. However, purified MCO266 exhibited Mn(II)-oxidizing activity when combined with cell outer membrane component (COMC) fractions in vitro. We demonstrated that Mn(II) oxidation and aggregate formation occurred through an oxygen-dependent biotic transformation process that requires a certain minimum Mn(II) concentration. We propose an approximate electron transfer pathway in which MCO266 transfers only one electron to convert Mn(II) to Mn(III) and then cooperates with other COMC electron transporters to transfer the other electron required to oxidize Mn(III) to Mn(IV).

Simulation of the cavity-binding site of three bacterial multicopper oxidases upon complex stabilization: interactional profile and electron transference pathways

Previous studies have shown that multicopper oxidases (MCOs) oxidize organic and inorganic compounds through oxidation–reduction reactions in which three structurally and functionally arranged copper centers coordinate the uptake of an electron from a reduced substrate. Structural comparisons among three bacterial MCOs, with high structural homology and available three-dimensional information, reveal that the primary structural differences between these MCOs are located near the mononuclear copper center (T1Cu), where substrate oxidation occurs, as opposed to where the reduction of oxygen to water occurs at the trinuclear center. Nevertheless, this substrate oxidation is achieved through an outer-sphere electron transfer mechanism that does not generate a stable substrate–enzyme complex. In this study, MCOs from Thermus thermophilus (Tth-MCO), Bacillus subtilis (CotA), and Escherichia coli (CueO), which have been previously determined through X-ray crystallography, were used as models to analyze the binding modes of these MCOs to three organic molecules, with specific interest in the substrate-binding site. The binding mode of the electron-donor molecule to the electron transfer binding site was primarily attributed to hydrophobic contacts, which likely play an important role in the determination of substrate specificity. Some complexes generated in this study showed an electron donor molecule conformation in which an electron could be directly transferred to the histidines coordinating T1Cu, while for others additional electron transference pathways were also possible through the participation of charged residues during electron transfer.

Mn(II,III) oxidation and MnO 2 mineralization by an expressed bacterial multicopper oxidase

Reactive Mn(IV) oxide minerals are ubiquitous in the environment and control the bioavailability and distribution of many toxic and essential elements and organic compounds. Their formation is thought to be dependent on microbial enzymes, because spontaneous Mn(II) to Mn(IV) oxidation is slow. Several species of marine Bacillus spores oxidize Mn(II) on their exosporium, the outermost layer of the spore, encrusting them with Mn(IV) oxides. Molecular studies have identified the mnx (Mn oxidation) genes, including mnxG, encoding a putative multicopper oxidase (MCO), as responsible for this two-electron oxidation, a surprising finding because MCOs only catalyze single-electron transfer reactions. Characterization of the enzymatic mechanism has been hindered by the lack of purified protein. By purifying active protein from the mnxDEFG expression construct, we found that the resulting enzyme is a blue (absorption maximum 590 nm) complex containing MnxE, MnxF, and MnxG proteins. Further, by analyzing the Mn(II)- and (III)-oxidizing activity in the presence of a Mn(III) chelator, pyrophosphate, we found that the complex facilitates both electron transfers from Mn(II) to Mn(III) and from Mn(III) to Mn(IV). X-ray absorption spectroscopy of the Mn mineral product confirmed its similarity to Mn(IV) oxides generated by whole spores. Our results demonstrate that Mn oxidation from soluble Mn(II) to Mn(IV) oxides is a two-step reaction catalyzed by an MCO-containing complex. With the purification of active Mn oxidase, we will be able to uncover its mechanism, broadening our understanding of Mn mineral formation and the bioinorganic capabilities of MCOs.

Bilirubin oxidase from Bacillus pumilus: A promising enzyme for the elaboration of efficient cathodes in biofuel cells

A CotA multicopper oxidase (MCO) from Bacillus pumilus, previously identified as a laccase, has been studied and characterized as a new bacterial bilirubin oxidase (BOD). The 59kDa protein containing four coppers, was successfully over-expressed in Escherichia coli and purified to homogeneity in one step. This 509 amino-acid enzyme, having 67% and 26% sequence identity with CotA from Bacillus subtilis and BOD from Myrothecium verrucaria, respectively, shows higher turnover activity towards bilirubin compared to other bacterial MCOs. The current density for O2 reduction, when immobilized in a redox hydrogel, is only 12% smaller than the current obtained with Trachyderma tsunodae BOD. Under continuous electrocatalysis, an electrode modified with the new BOD is more stable, and has a higher tolerance towards NaCl, than a T. tsunodae BOD modified electrode. This makes BOD from B. pumilus an attractive new candidate for application in biofuel cells (BFCs) and biosensors.

A bacterial laccase from marine microbial metagenome exhibiting chloride tolerance and dye decolorization ability

Laccases are blue multicopper oxidases with potential applications in environmental and industrial biotechnology. In this study, a new bacterial laccase gene of 1.32 kb was obtained from a marine microbial metagenome of the South China Sea by using a sequence screening strategy. The protein (named as Lac15) of 439 amino acids encoded by the gene contains three conserved Cu(2+)-binding domains, but shares less than 40% of sequence identities with all of the bacterial multicopper oxidases characterized. Lac15, recombinantly expressed in Escherichia coli, showed high activity towards syringaldazine at pH 6.5-9.0 with an optimum pH of 7.5 and with the highest activity occurring at 45 °C. Lac15 was stable at pH ranging from 5.5 to 9.0 and at temperatures from 15 to 45 °C. Distinguished from fungal laccases, the activity of Lac15 was enhanced twofold by chloride at concentrations lower than 700 mM, and kept the original level even at 1,000 mM chloride. Furthermore, Lac15 showed an ability to decolorize several industrial dyes of reactive azo class under alkalescent conditions. The properties of alkalescence-dependent activity, high chloride tolerance, and dye decolorization ability make the new laccase Lac15 an alternative for specific industrial applications.

Surface display of heterologous proteins in Bacillus thuringiensis using a peptidoglycan hydrolase anchor

BackgroundPrevious studies have revealed that the lysin motif (LysM) domains of bacterial cell wall-degrading enzymes are able to bind to peptidoglycan moieties of the cell wall. This suggests an approach for a cell surface display system in Gram-positive bacteria using a LysM-containing protein as the anchoring motif. In this study, we developed a new surface display system in B. thuringiensis using a LysM-containing peptidoglycan hydrolase, endo-β-N-acetylglucosaminidase (Mbg), as the anchor protein.ResultsHomology searching in the B. thuringiensis YBT-1520 genome revealed a putative peptidoglycan hydrolase gene. The encoded protein, Mbg, exhibited substantial cell-wall binding capacity. The deduced amino acid sequence of Mbg was structurally distinguished as an N-terminal domain with two tandemly aligned LysMs and a C-terminal catalytic domain. A GFP-fusion protein was expressed and used to verify the surface localization by Western blot, flow cytometry, protease accessibility, SDS sensitivity, immunofluorescence, and electron microscopy assays. Low-level constitutive expression of Mbg was elevated by introducing a sporulation-independent promoter of cry3Aa. Truncated Mbg domains with separate N-terminus (Mbgn), C-terminus (Mbgc), LysM1, or LysM2 were further compared for their cell-wall displaying efficiencies. The Mbgn moiety contributed to cell-wall anchoring, while LysM1 was the active domain. Two tandemly repeated Mbgns exhibited the highest display activity, while the activity of three repeated Mbgns was decreased. A heterologous bacterial multicopper oxidase (WlacD) was successfully displayed onto the surface of B. thuringiensis target cells using the optimum (Mbgn)2 anchor, without radically altering its catalytic activity.ConclusionMbg can be a functional anchor protein to target different heterologous proteins onto the surface of B. thuringiensis cells. Since the LysM domain appears to be universal in Gram-positive bacteria, the strategy presented here could be applicable in other bacteria for developing this type of system.

Direct Heterogeneous Electron Transfer Reactions of Bacillus halodurans Bacterial Blue Multicopper Oxidase

AbstractDirect electron transfer reactions of Bacillus halodurans bacterial multicopper oxidase on bare spectrographic graphite, as well as bare and thiol‐modified gold electrodes were studied using cyclic voltammetry, potentiometry, amperometry, and spectroelectrochemistry. The redox potential of the T1 site of the enzyme was measured using mediatorless redox titration and found to be 325 mV±10 mV vs. NHE. From measurements with a mercaptopropionic acid‐modified gold electrode under aerobic conditions a midpoint potential of 360 mV vs. NHE for the T2/T3 copper cluster is deduced. Differing from most other characterized laccases of fungal and plant origins this bacterial enzyme exhibits bioelectrocatalytic activity at neutral pH and tolerates high chloride concentrations (200 mM), conditions that usually strongly inhibit catalysis. Moreover, it has the very high affinity towards molecular oxygen both in solution and in the adsorbed state (KM≤50 μM).