The "life-span" of lytic polysaccharide monooxygenases (LPMOs) correlates to the number of turnovers in the reductant peroxidase reaction.

Lytic polysaccharide monooxygenases (LPMOs) are monocopper enzymes that degrade the insoluble crystalline polysaccharides cellulose and chitin. Besides the H2O2 cosubstrate, the cleavage of glycosidic bonds by LPMOs depends on the presence of a reductant needed to bring the enzyme into its reduced, catalytically active Cu(I) state. Reduced LPMOs that are not bound to substrate catalyze reductant peroxidase reactions, which may lead to oxidative damage and irreversible inactivation of the enzyme. However, the kinetics of this reaction remain largely unknown, as do possible variations between LPMOs belonging to different families. Here, we describe the kinetic characterization of two fungal family AA9 LPMOs, TrAA9A of Trichoderma reesei and NcAA9C of Neurospora crassa, and two bacterial AA10 LPMOs, ScAA10C of Streptomyces coelicolor and SmAA10A of Serratia marcescens. We found peroxidation of ascorbic acid and methyl-hydroquinone resulted in the same probability of LPMO inactivation (pi), suggesting that inactivation is independent of the nature of the reductant. We showed the fungal enzymes were clearly more resistant toward inactivation, having pi values of less than 0.01, whereas the pi for SmAA10A was an order of magnitude higher. However, the fungal enzymes also showed higher catalytic efficiencies (kcat/KM(H2O2)) for the reductant peroxidase reaction. This inverse linear correlation between the kcat/KM(H2O2) and pi suggests that, although having different life spans in terms of the number of turnovers in the reductant peroxidase reaction, LPMOs that are not bound to substrates have similar half-lives. These findings have not only potential biological but also industrial implications.

Kinetics of H2O2-driven catalysis by a lytic polysaccharide monooxygenase from the fungus Trichoderma reesei.

Owing to their ability to break glycosidic bonds in recalcitrant crystalline polysaccharides such as cellulose, the catalysis effected by lytic polysaccharide monooxygenases (LPMOs) is of major interest. Kinetics of these reductant-dependent, monocopper enzymes is complicated by the insoluble nature of the cellulose substrate and parallel, enzyme-dependent, and enzyme-independent side reactions between the reductant and oxygen-containing cosubstrates. Here, we provide kinetic characterization of cellulose peroxygenase (oxidative cleavage of glycosidic bonds in cellulose) and reductant peroxidase (oxidation of the reductant) activities of the LPMO TrAA9A of the cellulose-degrading model fungus Trichoderma reesei. The catalytic efficiency [Formula: see text] of the cellulose peroxygenase reaction (kcat = 8.5 s-1, and [Formula: see text] ) was an order of magnitude higher than that of the reductant (ascorbic acid) peroxidase reaction. The turnover of H2O2 in the ascorbic acid peroxidase reaction followed the ping-pong mechanism and led to irreversible inactivation of the enzyme with a probability of 0.0072. Using theoretical analysis, we suggest a relationship between the half-life of LPMO, the values of kinetic parameters, and the concentrations of the reactants.

Kinetic insights into the role of the reductant in H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase.

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 (KmRapp). Theoretical analyses revealed that KmRapp 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.

Kinetics of H2O2-driven degradation of chitin by a bacterial lytic polysaccharide monooxygenase.

Lytic polysaccharide monooxygenases (LPMOs) catalyze the oxidative cleavage of glycosidic bonds in recalcitrant polysaccharides, such as cellulose and chitin, and are of interest in biotechnological utilization of these abundant biomaterials. It has recently been shown that LPMOs can use H2O2, instead of O2, as a cosubstrate. This peroxygenase-like reaction by a monocopper enzyme is unprecedented in nature and opens new avenues in chemistry and enzymology. Here, we provide the first detailed kinetic characterization of chitin degradation by the bacterial LPMO chitin-binding protein CBP21 using H2O2 as cosubstrate. The use of 14C-labeled chitin provided convenient and sensitive detection of the released soluble products, which enabled detailed kinetic measurements. The kcat for chitin oxidation found here (5.6 s-1) is more than an order of magnitude higher than previously reported (apparent) rate constants for reactions containing O2 but no added H2O2 The kcat/Km for H2O2-driven degradation of chitin was on the order of 106 m-1 s-1, indicating that LPMOs have catalytic efficiencies similar to those of peroxygenases. Of note, H2O2 also inactivated CBP21, but the second-order rate constant for inactivation was about 3 orders of magnitude lower than that for catalysis. In light of the observed CBP21 inactivation at higher H2O2 levels, we conclude that controlled generation of H2O2in situ seems most optimal for fueling LPMO-catalyzed oxidation of polysaccharides.

When substrate inhibits and inhibitor activates: implications of ß-glucosidases.

BACKGROUND: ß-glucosidases (BGs) catalyze the hydrolysis of ß-glycosidic bonds in glucose derivatives. They constitute an important group of enzymes with biotechnological interest like supporting cellulases in degradation of lignocellulose to fermentable sugars. In the latter context, the glucose tolerant BGs are of particular interest. These BGs often show peculiar kinetics, including inhibitory effects of substrates and activating effects of inhibitors, such as glucose or xylose. The mechanisms behind the activating/inhibiting effects are poorly understood. The nonproductive binding of substrate is expected in cases where enzymes with multiple consecutive binding subsites are studied on substrates with a low degree of polymerization. The effects of inhibitors to BGs exerting nonproductive binding of substrate have not been discussed in the literature before. RESULTS: Here, we performed analyses of different reaction schemes using the catalysis by retaining BGs as a model. We found that simple competition of inhibitor with nonproductive binding of substrate can account for the activation of enzyme by inhibitor without involving any allosteric effects. The transglycosylation to inhibitor was also able to explain the activating effect of inhibitor. For both mechanisms, the activation was caused by the increase of kcat with increasing inhibitor concentration, while kcat/Km always decreased. Therefore, the activation by inhibitor was more pronounced at high substrate concentrations. The possible contribution of the two mechanisms in the activation by inhibitor was dependent on the rate-limiting step of glycosidic bond hydrolysis as well as on whether and which glucose-unit-binding subsites are interacting. CONCLUSION: Knowledge on the mechanisms of the activating/inhibiting effects of inhibitors helps the rational engineering and selection of BGs for biotechnological applications. Provided that the catalysis is consistent with the reaction schemes addressed here and underlying assumptions, the mechanism of activation by inhibitor reported here is applicable for all enzymes exerting nonproductive binding of substrate.

Human Chitotriosidase Is an Endo-Processive Enzyme.

Human chitotriosidase (HCHT) is involved in immune response to chitin-containing pathogens in humans. The enzyme is able to degrade chitooligosaccharides as well as crystalline chitin. The catalytic domain of HCHT is connected to the carbohydrate binding module (CBM) through a flexible hinge region. In humans, two active isoforms of HCHT are found-the full length enzyme and its truncated version lacking CBM and the hinge region. The active site architecture of HCHT is reminiscent to that of the reducing-end exo-acting processive chitinase ChiA from bacterium Serratia marcescens (SmChiA). However, the presence of flexible hinge region and occurrence of two active isoforms are reminiscent to that of non-processive endo-chitinase from S. marcescens, SmChiC. Although the studies on soluble chitin derivatives suggest the endo-character of HCHT, the mode of action of the enzyme on crystalline chitin is not known. Here, we made a thorough characterization of HCHT in terms of the mode of action, processivity, binding, and rate constants for the catalysis and dissociation using α-chitin as substrate. HCHT efficiently released the end-label from reducing-end labelled chitin and had also high probability (95%) of endo-mode initiation of processive run. These results qualify HCHT as an endo-processive enzyme. Processivity and the rate constant of dissociation of HCHT were found to be in-between those, characteristic to processive exo-enzymes, like SmChiA and randomly acting non-processive endo-enzymes, like SmChiC. Apart from increasing the affinity for chitin, CBM had no major effect on kinetic properties of HCHT.

Slow Off-rates and Strong Product Binding Are Required for Processivity and Efficient Degradation of Recalcitrant Chitin by Family 18 Chitinases.

Processive glycoside hydrolases are the key components of enzymatic machineries that decompose recalcitrant polysaccharides, such as chitin and cellulose. The intrinsic processivity (P(Intr)) of cellulases has been shown to be governed by the rate constant of dissociation from polymer chain (koff). However, the reported koff values of cellulases are strongly dependent on the method used for their measurement. Here, we developed a new method for determining koff, based on measuring the exchange rate of the enzyme between a non-labeled and a (14)C-labeled polymeric substrate. The method was applied to the study of the processive chitinase ChiA from Serratia marcescens. In parallel, ChiA variants with weaker binding of the N-acetylglucosamine unit either in substrate-binding site -3 (ChiA-W167A) or the product-binding site +1 (ChiA-W275A) were studied. Both ChiA variants showed increased off-rates and lower apparent processivity on α-chitin. The rate of the production of insoluble reducing groups on the reduced α-chitin was an order of magnitude higher than koff, suggesting that the enzyme can initiate several processive runs without leaving the substrate. On crystalline chitin, the general activity of the wild type enzyme was higher, and the difference was magnifying with hydrolysis time. On amorphous chitin, the variants clearly outperformed the wild type. A model is proposed whereby strong interactions with polymer in the substrate-binding sites (low off-rates) and strong binding of the product in the product-binding sites (high pushing potential) are required for the removal of obstacles, like disintegration of chitin microfibrils.

The predominant molecular state of bound enzyme determines the strength and type of product inhibition in the hydrolysis of recalcitrant polysaccharides by processive enzymes.

Processive enzymes are major components of the efficient enzyme systems that are responsible for the degradation of the recalcitrant polysaccharides cellulose and chitin. Despite intensive research, there is no consensus on which step is rate-limiting for these enzymes. Here, we performed a comparative study of two well characterized enzymes, the cellobiohydrolase Cel7A from Hypocrea jecorina and the chitinase ChiA from Serratia marcescens. Both enzymes were inhibited by their disaccharide product, namely chitobiose for ChiA and cellobiose for Cel7A. The products behaved as noncompetitive inhibitors according to studies using the (14)C-labeled crystalline polymeric substrates (14)C chitin nanowhiskers and (14)C-labeled bacterial microcrystalline cellulose for ChiA and Cel7A, respectively. The resulting observed Ki (obs) values were 0.45 ± 0.08 mm for ChiA and 0.17 ± 0.02 mm for Cel7A. However, in contrast to ChiA, the Ki (obs) of Cel7A was an order of magnitude higher than the true Ki value governed by the thermodynamic stability of the enzyme-inhibitor complex. Theoretical analysis of product inhibition suggested that the inhibition strength and pattern can be accounted for by assuming different rate-limiting steps for ChiA and Cel7A. Measuring the population of enzymes whose active site was occupied by a polymer chain revealed that Cel7A was bound predominantly via its active site. Conversely, the active-site-mediated binding of ChiA was slow, and most ChiA exhibited a free active site, even when the substrate concentration was saturating for the activity. Collectively, our data suggest that complexation with the polymer chain is rate-limiting for ChiA, whereas Cel7A is limited by dissociation.

The helicase CaHmi1p is required for wild-type mitochondrial DNA organization in Candida albicans.

The mechanistic details of mtDNA maintenance in petite-negative yeasts have remained largely unexplored. We report here that the DNA helicase Hmi1p plays a crucial role in mtDNA stability in Candida albicans. Like its counterpart in Saccharomyces cerevisiae, Hmi1p in C. albicans (CaHmi1p) contains a C-terminal mitochondrial targeting signal that is functional in both organisms. Biochemical analysis demonstrates that CaHmi1p is a protein possessing ATP-dependent 3'-5' DNA-unwinding activity. Deletion of both HMI1 alleles does not lead to complete loss of mtDNA in C. albicans; however, substantial fragmentation of the wild-type mitochondrial genome, reduction of mtDNA mass and loss of wild-type nucleoid distribution occur. Specific regions of the mitochondrial genome give rise to mtDNA molecule populations with altered characteristics upon CaHMI1 deletion. Fragmentation of the mitochondrial genome can be reversed by reintroduction of CaHmi1p. This is the first time that a gene required for wild-type mtDNA maintenance in S. cerevisiae has been demonstrated to be nonessential in a petite-negative yeast.

Hmi1p from Saccharomyces cerevisiae mitochondria is a structure-specific DNA helicase.

Hmi1p is a Saccharomyces cerevisiae mitochondrial DNA helicase that is essential for the maintenance of functional mitochondrial DNA. Hmi1p belongs to the superfamily 1 of helicases and is a close homologue of bacterial PcrA and Rep helicases. We have overexpressed and purified recombinant Hmi1p from Escherichia coli and describe here the biochemical characteristics of its DNA helicase activities. Among nucleotide cofactors, the DNA unwinding by Hmi1p was found to occur efficiently only in the presence of ATP and dATP. Hmi1p could unwind only the DNA substrates with a 3'-single-stranded overhang. The length of the 3'-overhang needed for efficient targeting of the helicase to the substrate depended on the substrate structure. For substrates consisting of duplex DNA with a 3'-single-stranded DNA overhang, at least a 19-nt 3'-overhang was needed. In the case of forked substrates with both 3'- and 5'-overhangs, a 9-nt 3'-overhang was sufficient provided that the 5'-overhang was also 9 nt in length. In flap-structured substrates mimicking the chain displacement structures in DNA recombination process, only a 5-nt 3'-single-stranded DNA tail was required for efficient unwinding by Hmi1p. These data indicate that Hmi1p may be targeted to a specific 3'-flap structure, suggesting its possible role in DNA recombination.

Helicase Hmi1 stimulates the synthesis of concatemeric mitochondrial DNA molecules in yeast Saccharomyces cerevisiae.

Hmi1p is a helicase in the yeast Saccharomyces cerevisiae required for maintenance of the wild-type mitochondrial genome. Disruption of the HMI1 ORF generates rho(-) and rho(0) cells. Here we demonstrate that, in rho(-) yeast strains, Hmi1p stimulates the synthesis of long concatemeric mitochondrial DNA molecules associated with a reduction in the number of nucleoids used for mitochondrial DNA packaging. Surprisingly, the ATPase negative mutants of Hmi1p can also stimulate the synthesis of long concatemeric rho(-) mitochondrial DNA molecules and support the maintenance of the wild-type mitochondrial genome, albeit with reduced efficiency. We show that, in the mutant hmi1-5 background, the wild-type mitochondrial DNA is fragmented; and we propose that, in hmi1Delta yeast cells, the loss of the wild-type mitochondrial genome is caused by this fragmentation of the mitochondrial DNA.