Kinetic Characterization of a Putatively Chitin-Active LPMO Reveals a Preference for Soluble Substrates and Absence of Monooxygenase Activity.
Ontology highlight
ABSTRACT:
SUBMITTER: Rieder L
PROVIDER: S-EPMC8453653 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:The potentially tridentate ligand bis[(1-methyl-2-benzimidazolyl)ethyl]amine (2BB) was employed to prepare copper complexes [(2BB)CuI]OTf and [(2BB)CuII(H2O)2](OTf)2 as bioinspired models of lytic polysaccharide copper-dependent monooxygenase (LPMO) enzymes. Solid-state characterization of [(2BB)CuI]OTf revealed a Cu(I) center with a T-shaped coordination environment and metric parameters in the range of those observed in reduced LPMOs. Solution characterization of [(2BB)CuII(H2O)2](OTf)2 indicates that [(2BB)CuII(H2O)2]2+ is the main species from pH 4 to 7.5; above pH 7.5, the hydroxo-bridged species [{(2BB)CuII(H2O) x }2(μ-OH)2]2+ is also present, on the basis of cyclic voltammetry and mass spectrometry. These observations imply that deprotonation of the central amine of Cu(II)-coordinated 2BB is precluded, and by extension, amine deprotonation in the histidine brace of LPMOs appears unlikely at neutral pH. The complexes [(2BB)CuI]OTf and [(2BB)CuII(H2O)2](OTf)2 act as precursors for the oxidative degradation of cellobiose as a cellulose model substrate. Spectroscopic and reactivity studies indicate that a dicopper(II) side-on peroxide complex generated from [(2BB)CuI]OTf/O2 or [(2BB)CuII(H2O)2](OTf)2/H2O2/NEt3 oxidizes cellobiose both in acetonitrile and aqueous phosphate buffer solutions, as evidenced from product analysis by high-performance liquid chromatography-mass spectrometry. The mixture of [(2BB)CuII(H2O)2](OTf)2/H2O2/NEt3 results in more extensive cellobiose degradation. Likewise, the use of both [(2BB)CuI]OTf and [(2BB)CuII(H2O)2](OTf)2 with KO2 afforded cellobiose oxidation products. In all cases, a common Cu(II) complex formulated as [(2BB)CuII(OH)(H2O)]+ was detected by mass spectrometry as the final form of the complex.
| S-EPMC6648734 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that catalyze oxidative cleavage of glycosidic bonds in polysaccharides in the presence of an external electron donor (reductant). In the classical O2-driven monooxygenase reaction, the reductant is needed in stoichiometric amounts. In a recently discovered, more efficient H2O2-driven reaction, the reductant would be needed only for the initial reduction (priming) of the LPMO to its catalytically active Cu(I) form. However, the influence of the reductant on reducing the LPMO or on H2O2 production in the reaction remains undefined. Here, we conducted a detailed kinetic characterization to investigate how the reductant affects H2O2-driven degradation of 14C-labeled chitin by a bacterial LPMO, SmLPMO10A (formerly CBP21). Sensitive detection of 14C-labeled products and careful experimental set-ups enabled discrimination between the effects of the reductant on LPMO priming and other effects, in particular enzyme-independent production of H2O2 through reactions with O2 When supplied with H2O2, SmLPMO10A catalyzed 18 oxidative cleavages per molecule of ascorbic acid, suggesting a "priming reduction" reaction. The dependence of initial rates of chitin degradation on reductant concentration followed hyperbolic saturation kinetics, and differences between the reductants were manifested in large variations in their half-saturating concentrations (K mR app). Theoretical analyses revealed that K mR app decreases with a decreasing rate of polysaccharide-independent LPMO reoxidation (by either O2 or H2O2). We conclude that the efficiency of LPMO priming depends on the relative contributions of reductant reactivity, on the LPMO's polysaccharide monooxygenase/peroxygenase and reductant oxidase/peroxidase activities, and on reaction conditions, such as O2, H2O2, and polysaccharide concentrations.
| S-EPMC6364757 | biostudies-literature
Project description:The controlled oxidation of methane to methanol is a chemical transformation of great value, particularly in the pursuit of alternative fuels, but the reaction remains underutilized industrially because of inefficient and costly synthetic procedures. In contrast, methane monooxygenase enzymes (MMOs) from methanotrophic bacteria achieve this chemistry efficiently under ambient conditions. In this Account, we discuss the first observable step in the oxidation of methane at the carboxylate-bridged diiron active site of the soluble MMO (sMMO), namely, the reductive activation of atmospheric O(2). The results provide benchmarks against which the dioxygen activation mechanisms of other bacterial multicomponent monooxygenases can be measured. Molecular oxygen reacts rapidly with the reduced diiron(II) cen-ter of the hydroxylase component of sMMO (MMOH). The first spectroscopically characterized intermediate that results from this process is a peroxodiiron(III) species, P*, in which the iron atoms have identical environments. P* converts to a second peroxodiiron(III) unit, H(peroxo), in a process accompanied by the transfer of a proton, probably with the assistance of a residue near the active site. Proton-promoted O-O bond scission and rearrangement of the diiron core then leads to a diiron(IV) unit, termed Q, that is directly responsible for the oxidation of methane to methanol. In one section of this Account, we provide a detailed discussion of these processes, with particular emphasis on possible structures of the intermediates. The geometries of P* and H(peroxo) are currently unknown, and recent synthetic modeling chemistry has highlighted the need for further structural characterization of Q, currently assigned as a di(μ-oxo)diiron(IV) "diamond core." In another section of the Account, we discuss in detail proton transfer during the O(2) activation events. The role of protons in promoting O-O bond cleavage, thereby initiating the conversion of H(peroxo) to Q, was previously a controversial topic. Recent studies of the mechanism, covering a range of pH values and in D(2)O instead of H(2)O, confirmed conclusively that the transfer of protons, possibly at or near the active site, is necessary for both P*-to-H(peroxo) and H(peroxo)-to-Q conversions. Specific mechanistic insights into these processes are provided. In the final section of the Account, we present our view of experiments that need to be done to further define crucial aspects of sMMO chemistry. Here our goal is to detail the challenges that we and others face in this research, particularly with respect to some long-standing questions about the system, as well as approaches that might be used to solve them.
| S-EPMC3079780 | biostudies-literature
Project description:Lytic polysaccharide monooxygenases (LPMOs) have a unique ability to activate molecular oxygen for subsequent oxidative cleavage of glycosidic bonds. To provide insight into the mode of action of these industrially important enzymes, we have performed an integrated NMR/electron paramagnetic resonance (EPR) study into the detailed aspects of an AA10 LPMO-substrate interaction. Using NMR spectroscopy, we have elucidated the solution-phase structure of apo-BlLPMO10A from Bacillus licheniformis, along with solution-phase structural characterization of the Cu(I)-LPMO, showing that the presence of the metal has minimal effects on the overall protein structure. We have, moreover, used paramagnetic relaxation enhancement (PRE) to characterize Cu(II)-LPMO by NMR spectroscopy. In addition, a multifrequency continuous-wave (CW)-EPR and 15N-HYSCORE spectroscopy study on the uniformly isotope-labeled 63Cu(II)-bound 15N-BlLPMO10A along with its natural abundance isotopologue determined copper spin-Hamiltonian parameters for LPMOs to markedly improved accuracy. The data demonstrate that large changes in the Cu(II) spin-Hamiltonian parameters are induced upon binding of the substrate. These changes arise from a rearrangement of the copper coordination sphere from a five-coordinate distorted square pyramid to one which is four-coordinate near-square planar. There is also a small reduction in metal-ligand covalency and an attendant increase in the d(x2-y2) character/energy of the singly occupied molecular orbital (SOMO), which we propose from density functional theory (DFT) calculations predisposes the copper active site for the formation of a stable Cu-O2 intermediate. This switch in orbital character upon addition of chitin provides a basis for understanding the coupling of substrate binding with O2 activation in chitin-active AA10 LPMOs.
| S-EPMC7431007 | biostudies-literature
Project description:Engineered tissues require enhanced organization of cells and extracellular matrix (ECM) for proper function. To promote cell organization, substrates with controlled micro- and nanopatterns have been developed as supports for cell growth, and to induce cellular elongation and orientation via contact guidance. Micropatterned ultra-thin biodegradable substrates are desirable for implantation in the host tissue. These substrates, however, need to be mechanically robust to provide substantial support for the generation of new tissues, to be easily retrievable, and to maintain proper handling characteristics. Here, we introduce ultra-thin (<10 ?m), self-assembled chitin nanofiber substrates micropatterned with replica molding for engineering cell sheets. These substrates are biodegradable, mechanically strong, yet flexible, and easily manipulated into the desired shape. As a proof-of-concept, fibroblast cell proliferation, elongation, and alignment were studied on the developed substrates with different pattern dimensions. On the optimized substrates, the majority of the cells aligned (<10°) along the major axis of micropatterned features. With the ease of fabrication and mechanical robustness, the substrates presented herein can be utilized as versatile system for the engineering and delivery of ordered tissue in applications such as myocardial repair.
| S-EPMC3809896 | biostudies-literature
Project description:The hydroxylation or epoxidation of hydrocarbons by bacterial multicomponent monooxygenases (BMMs) requires the interplay of three or four protein components. How component protein interactions control catalysis, however, is not well understood. In particular, the binding sites of the reductase components on the surface of their cognate hydroxylases and the role(s) that the regulatory proteins play during intermolecular electron transfer leading to the hydroxylase reduction have been enigmatic. Here we determine the reductase binding site on the hydroxylase of a BMM enzyme, soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath). We present evidence that the ferredoxin domain of the reductase binds to the canyon region of the hydroxylase, previously determined to be the regulatory protein binding site as well. The latter thus inhibits reductase binding to the hydroxylase and, consequently, intermolecular electron transfer from the reductase to the hydroxylase diiron active site. The binding competition between the regulatory protein and the reductase may serve as a control mechanism for regulating electron transfer, and other BMM enzymes are likely to adopt the same mechanism.
| S-EPMC4105053 | biostudies-literature
Project description:Members of the antibiotic biosynthesis monooxygenase family catalyze O2-dependent oxidations and oxygenations in the absence of any metallo- or organic cofactor. How these enzymes surmount the kinetic barrier to reactions between singlet substrates and triplet O2 is unclear, but the reactions have been proposed to occur via a flavin-like mechanism, where the substrate acts in lieu of a flavin cofactor. To test this model, we monitored the uncatalyzed and enzymatic reactions of dithranol, a substrate for the nogalamycin monooxygenase (NMO) from Streptomyces nogalater As with flavin, dithranol oxidation was faster at a higher pH, although the reaction did not appear to be base-catalyzed. Rather, conserved asparagines contributed to suppression of the substrate pKa The same residues were critical for enzymatic catalysis that, consistent with the flavoenzyme model, occurred via an O2-dependent slow step. Evidence for a superoxide/substrate radical pair intermediate came from detection of enzyme-bound superoxide during turnover. Small molecule and enzymatic superoxide traps suppressed formation of the oxygenation product under uncatalyzed conditions, whereas only the small molecule trap had an effect in the presence of NMO. This suggested that NMO both accelerated the formation and directed the recombination of a superoxide/dithranyl radical pair. These catalytic strategies are in some ways flavin-like and stand in contrast to the mechanisms of urate oxidase and (1H)-3-hydroxy-4-oxoquinaldine 2,4-dioxygenase, both cofactor-independent enzymes that surmount the barriers to direct substrate/O2 reactivity via markedly different means.
| S-EPMC5016173 | biostudies-literature
Project description:Physiochemical methods have generally been used to "open-up" biomass substrates/pulps and have been the main method used to fibrillate cellulose. However, recent work has shown that canonical cellulase enzymes such as endoglucanases, in combination with "amorphogenesis inducing" proteins such as lytic polysaccharide monooxygenases (LPMO), swollenin and hemicellulases, are able to increase cellulose accessibility. In the work reported here different combinations of endoglucanase, LPMO and xylanase were applied to Kraft pulps to assess their potential to induce fibrillation at low enzyme loading over a short time period. Although gross fiber properties (fiber length, width and morphology) were relatively unchanged, over a short period of time, the intrinsic physicochemical characteristics of the pulp fibers (e.g. cellulose accessibility/DP/crystallinity/charge) were positively enhanced by the synergistic cooperation of the enzymes. LPMO addition resulted in the oxidative cleavage of the pulps, increasing the negative charge (~100?mmol kg-1) on the cellulose fibers. This improved cellulose nanofibrilliation while stabilizing the nanofibril suspension (zeta potential ??=?~60?mV), without sacrificing nanocellulose thermostability. The combination of endoglucanase, LPMO and xylanases was shown to facilitate nanofibrillation, potentially reducing the need for mechanical refining while resulting in a pulp with a more uniform nanofibril composition.
| S-EPMC5816652 | biostudies-literature
Project description:The presented data are related to the research article entitled "Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates" by Ozgur et al. (2017) [1]. This data report describes the challenges of investigating the concentration-dependent transport of P-glycoprotein (P-gp) substrates with relatively low aqueous solubility. Thus, we provide solubility data on two prototypical P-gp substrates, digoxin and rhodamine 123, representing P-gp substrates with a relatively low- and high-aqueous solubility, respectively. We present a hypothetical Michaelis-Menten curve of the P-gp mediated transport of digoxin to demonstrate that the maximal donor concentration, which can be reached in the experimental transport buffer, is too low to yield transport data in the saturable range of the Michaelis-Menten relationship. Furthermore, we present data on the bidirectional transport of digoxin and rhodamine 123 across cell monolayers of the MDCK II MDR1 cell line and iP-pg cell line in the presence of the selective P-gp inhibitor, zosuquidar/LY335979.
| S-EPMC5848149 | biostudies-literature
Project description:The accumulation of chlorophenols (CPs) in the environment, due to their wide use as agrochemicals, has become a serious environmental problem. These organic halides can be degraded by aerobic microorganisms, where the initial steps of various biodegradation pathways include an oxidative dechlorinating process in which chloride is replaced by a hydroxyl substituent. Harnessing these dechlorinating processes could provide an opportunity for environmental remediation, but detailed catalytic mechanisms for these enzymes are not yet known. To close this gap, we now report transient kinetics and product analysis of the dechlorinating flavin-dependent monooxygenase, HadA, from the aerobic organism Ralstonia pickettii DTP0602, identifying several mechanistic properties that differ from other enzymes in the same class. We first overexpressed and purified HadA to homogeneity. Analyses of the products from single and multiple turnover reactions demonstrated that HadA prefers 4-CP and 2-CP over CPs with multiple substituents. Stopped-flow and rapid-quench flow experiments of HadA with 4-CP show the involvement of specific intermediates (C4a-hydroperoxy-FAD and C4a-hydroxy-FAD) in the reaction, define rate constants and the order of substrate binding, and demonstrate that the hydroxylation step occurs prior to chloride elimination. The data also identify the non-productive and productive paths of the HadA reactions and demonstrate that product formation is the rate-limiting step. This is the first elucidation of the kinetic mechanism of a two-component flavin-dependent monooxygenase that can catalyze oxidative dechlorination of various CPs, and as such it will serve as the basis for future investigation of enzyme variants that will be useful for applications in detoxifying chemicals hazardous to human health.
| S-EPMC5377798 | biostudies-literature