Christophe V. F. P. Laurent

Direct bioelectrocatalysis applied in biosensors, biofuel cells, and bioelectrosynthesis is based on an efficient electron transfer between enzymes and electrodes in the absence of redox mediators. Some oxidoreductases are capable of direct electron transfer (DET), while others achieve the enzyme to electrode electron transfer (ET) by employing an electron-transferring domain. Cellobiose dehydrogenase (CDH) is the most-studied multidomain bioelectrocatalyst and features a catalytic flavodehydrogenase domain and a mobile, electron-transferring cytochrome domain connected by a flexible linker. The ET to the physiological redox partner lytic polysaccharide monooxygenase or, ex vivo, electrodes depends on the flexibility of the electron transferring domain and its connecting linker, but the regulatory mechanism is little understood. Studying the linker sequences of currently characterized CDH classes we observed that the inner, mobile linker sequence is flanked by two outer linker regions that are in close contact with the adjacent domain. A function-based definition of the linker region in CDH is proposed and has been verified by rationally designed variants of Neurospora crassa CDH. The effect of linker length and its domain attachment on electron transfer rates has been determined by biochemical and electrochemical methods, while distances between the domains of CDH variants were computed. This study elucidates the regulatory mechanism of the interdomain linker on electron transfer by determining the minimum linker length, observing the effects of elongated linkers, and testing the covalent stabilization of a linker part to the flavodehydrogenase domain. The evolutionary guided, rational design of the interdomain linker provides a strategy to optimize electron transfer rates in multidomain enzymes and maximize their bioelectrocatalytic performance.

Copper-dependent lytic polysaccharide monooxygenases (LPMOs) classified in Auxiliary Activity (AA) families are considered indispensable as synergistic partners for cellulolytic enzymes to saccharify recalcitrant lignocellulosic plant biomass. In this study, we characterized two fungal oxidoreductases from the new AA16 family. We found that MtAA16A from Myceliophthora thermophila and AnAA16A from Aspergillus nidulans did not catalyze the oxidative cleavage of oligo- and polysaccharides. Indeed, the MtAA16A crystal structure showed a fairly LPMO-typical histidine brace active site, but the cellulose-acting LPMO-typical flat aromatic surface parallel to the histidine brace region was lacking. Further, we showed that both AA16 proteins are able to oxidize low-molecular-weight reductants to produce H₂O₂. The oxidase activity of the AA16s substantially boosted cellulose degradation by four AA9 LPMOs from M. thermophila (MtLPMO9s) but not by three AA9 LPMOs from Neurospora crassa (NcLPMO9s). The interplay with MtLPMO9s is explained by the H₂O₂-producing capability of the AA16s, which, in the presence of cellulose, allows the MtLPMO9s to optimally drive their peroxygenase activity. Replacement of MtAA16A by glucose oxidase (AnGOX) with the same H₂O₂-producing activity could only achieve less than 50% of the boosting effect achieved by MtAA16A, and earlier MtLPMO9B inactivation (6 h) was observed. To explain these results, we hypothesized that the delivery of AA16-produced H₂O₂ to the MtLPMO9s is facilitated by protein-protein interaction. Our findings provide new insights into the functions of copper-dependent enzymes and contribute to a further understanding of the interplay of oxidative enzymes within fungal systems to degrade lignocellulose.

An "imaginary transition structure" overlays the molecular graphs of the educt and product sides of an elementary chemical reaction in a single graph to highlight the changes in bond structure. We generalize this idea to reactions with complex mechanisms in a formally rigorous approach based on composing arrow-pushing steps represented as graph-transformation rules to construct an overall composite rule and a derived transition structure. This transition structure retains information about transient bond changes that are invisible at the overall level and can be constructed automatically from an existing database of detailed enzymatic mechanisms. We use the construction to (i) illuminate the distribution of catalytic action across enzymes and substrates and (ii) to search in a large database for reactions of known or unknown mechanisms that are compatible with the mechanism captured by the constructed composite rule.

The typically low thermodynamic and kinetic stability of enzymes is a bottleneck for their application in industrial synthesis. Baeyer-Villiger monooxygenases, which oxidize ketones to lactones using aerial oxygen, among other activities, suffer particularly from these instabilities. Previous efforts in protein engineering have increased thermodynamic stability but at the price of decreased activity. Here, we solved this trade-off by introducing mutations in a cyclohexanone monooxygenase from Acinetobacter sp., guided by a combination of rational and structure-guided consensus approaches. We developed variants with improved activity (1.5- to 2.5-fold) and increased thermodynamic (+5 °C T _m) and kinetic stability (8-fold). Our analysis revealed a crucial position in the cofactor binding domain, responsible for an 11-fold increase in affinity to the flavin cofactor, and explained using MD simulations. This gain in affinity was compatible with other mutations. While our study focused on a particular model enzyme, previous studies indicate that these findings are plausibly applicable to other BVMOs, and possibly to other flavin-dependent monooxygenases. These new design principles can inform the development of industrially robust, flavin-dependent biocatalysts for various oxidations.

Reaction mechanisms are often presented as sequences of elementary steps, such as codified by arrow pushing. We propose an approach for representing such mechanisms using graph transformation. In this framework, each elementary step is a rule for modifying a molecular graph and a mechanism is a sequence of such rules. To generate a compact representation of a multi-step reaction, we compose the rules of individual steps into a composite rule, providing a rigorous and fully automated approach to coarse-graining. While the composite rule retains the graphical conditions necessary for the execution of a mechanism, it also records information about transient changes not visible by comparing educts and products. By projecting the rule onto a single "overlay graph", we generalize Fujita's idea of an Imaginary Transition Structure from elementary reactions to composite reactions. The utility of the overlay graph construct is exemplified in the context of enzyme-catalyzed reactions. In a first application, we exploit mechanistic information in the Mechanism and Catalytic Site Atlas to construct overlay graphs of hydrolase reactions listed in the database. These graphs point at a spectrum of catalytic entanglement of enzyme and substrate, de-emphasizing the notion of a singular catalyst in favor of a collection of catalytic sites that can be distributed across enzyme and substrate. In a second application, we deploy composite rules to search the Rhea database for reactions of known or unknown mechanism that are, in principle, compatible with the mechanisms implied by the composite rules. We believe this work adds to the utility of graph-transformation formalisms in representing and reasoning about chemistry in an automated yet insightful fashion.

Lytic polysaccharide monooxygenases (LPMOs) play a key role in enzymatic degradation of hard‐to‐convert polysaccharides, such as chitin and cellulose. It is widely accepted that LPMOs catalyze a single regioselective oxidation of the C1 or C4 carbon of a glycosidic linkage, after which the destabilized linkage breaks. Here, a series of novel C4/C6 double oxidized cello‐oligosaccharides was discovered. Products were characterized, aided by sodium borodeuteride reduction and hydrophilic interaction chromatography coupled to mass spectrometric analysis. The C4/C6 double oxidized products were generated by C4 and C1/C4 oxidizing LPMOs, but not by C1 oxidizing ones. By performing incubation and reduction in H₂¹⁸O, it was confirmed that the C6 gem‐diol structure resulted from oxygenation, although oxidation to a C6 aldehyde, followed by hydration to the C6 gem‐diol, could not be excluded. These findings can be extended to how the reactive LPMO‐cosubstrate complex is positioned towards the substrate.

The design of enzymes is as challenging as it is consequential for making chemical synthesis in medical and industrial applications more efficient, cost-effective and environmentally friendly. While several aspects of this complex problem are computationally assisted, the drafting of catalytic mechanisms, i.e. the specification of the chemical steps—and hence intermediate states—that the enzyme is meant to implement, is largely left to human expertise. The ability to capture specific chemistries of multistep catalysis in a fashion that enables its computational construction and design is therefore highly desirable and would equally impact the elucidation of existing enzymatic reactions whose mechanisms are unknown. We use the mathematical framework of graph transformation to express the distinction between rules and reactions in chemistry. We derive about 1000 rules for amino acid side chain chemistry from the M-CSA database, a curated repository of enzymatic mechanisms. Using graph transformation, we are able to propose hundreds of hypothetical catalytic mechanisms for a large number of unrelated reactions in the Rhea database. We analyze these mechanisms to find that they combine in chemically sound fashion individual steps from a variety of known multistep mechanisms, showing that plausible novel mechanisms for catalysis can be constructed computationally. The source code of the initial prototype of our approach is available at https://github.com/Nojgaard/mechsearch Supplementary data are available at Bioinformatics online.

The natural function of cellobiose dehydrogenase (CDH) to donate electrons from its catalytic flavodehydrogenase (DH) domain via its cytochrome (CYT) domain to lytic polysaccharide monooxygenase (LPMO) is an example of a highly efficient extracellular electron transfer chain. To investigate the function of the CYT domain movement in the two occurring electron transfer steps, two CDHs from the ascomycete Neurospora crassa (NcCDHIIA and NcCDHIIB) and five chimeric CDH enzymes created by domain swapping were studied in combination with the fungus’ own LPMOs (NcLPMO9C and NcLPMO9F). Kinetic and electrochemical methods and hydrogen/deuterium exchange mass spectrometry were used to study the domain movement, interaction, and electron transfer kinetics. Molecular docking provided insights into the protein–protein interface, the orientation of domains, and binding energies. We find that the first, interdomain electron transfer step from the catalytic site in the DH domain to the CYT domain depends on steric and electrostatic interface complementarity and the length of the protein linker between both domains but not on the redox potential difference between the FAD and heme b cofactors. After CYT reduction, a conformational change of CDH from its closed state to an open state allows the second, interprotein electron transfer (IPET) step from CYT to LPMO to occur by direct interaction of the b-type heme and the type-2 copper center. Chimeric CDH enzymes favor the open state and achieve higher IPET rates by exposing the heme b cofactor to LPMO. The IPET, which is influenced by interface complementarity and the heme b redox potential, is very efficient with bimolecular rates between 2.9 × 10⁵ and 1.1 × 10⁶ M^–1 s^–1.

Background: Lytic polysaccharide monooxygenases (LPMOs) are powerful enzymes that oxidatively cleave plant cell wall polysaccharides. LPMOs classified as fungal Auxiliary Activities family 9 (AA9) have been mainly studied for their activity towards cellulose; however, various members of this AA9 family have been also shown to oxidatively cleave hemicelluloses, in particularly xyloglucan (XG). So far, it has not been studied in detail how various AA9 LPMOs act in XG degradation, and in particular, how the mode-of-action relates to the structural configuration of these LPMOs. Results: Two Neurospora crassa (Nc) LPMOs were found to represent different mode-of-action towards XG. Interestingly, the configuration of active site segments of these LPMOs differed as well, with a shorter Segment 1 (⁻Seg1) and a longer Segment 2 (⁺Seg2) present in NcLPMO9C and the opposite for NcLPMO9M (⁺Seg1⁻Seg2). We confirmed that NcLPMO9C cleaved the non-reducing end of unbranched glucosyl residues within XG via the oxidation of the C4-carbon. In contrast, we found that the oxidative cleavage of the XG backbone by NcLPMO9M occurred next to both unbranched and substituted glucosyl residues. The latter are decorated with xylosyl, xylosyl–galactosyl and xylosyl–galactosyl–fucosyl units. The relationship between active site segments and the mode-of-action of these NcLPMOs was rationalized by a structure-based phylogenetic analysis of fungal AA9 LPMOs. LPMOs with a ⁻Seg1⁺Seg2 configuration clustered together and appear to have a similar XG substitution-intolerant cleavage pattern. LPMOs with the ⁺Seg1⁻Seg2 configuration also clustered together and are reported to display a XG substitution-tolerant cleavage pattern. A third cluster contained LPMOs with a ⁻Seg1⁻Seg2 configuration and no oxidative XG activity. Conclusions: The detailed characterization of XG degradation products released by LPMOs reveal a correlation between the configuration of active site segments and mode-of-action of LPMOs. In particular, oxidative XG-active LPMOs, which are tolerant and intolerant to XG substitutions are structurally and phylogenetically distinguished from XG-inactive LPMOs. This study contributes to a better understanding of the structure–function relationship of AA9 LPMOs.