The rotamer of the second-sphere histidine in AA9 lytic polysaccharide monooxygenase is pH dependent.

Lytic polysaccharide monooxygenases (LPMOs) catalyze a reaction that is crucial for the biological decomposition of various biopolymers and for the industrial conversion of plant biomass. Despite the importance of LPMOs, the exact molecular-level nature of the reaction mechanism is still debated today. Here, we investigated the pH-dependent conformation of a second-sphere histidine (His) that we call the stacking histidine, which is conserved in fungal AA9 LPMOs and is speculated to assist catalysis in several of the LPMO reaction pathways.

Sourcing thermotolerant poly(ethylene terephthalate) hydrolase scaffolds from natural diversity.

Enzymatic deconstruction of poly(ethylene terephthalate) (PET) is under intense investigation, given the ability of hydrolase enzymes to depolymerize PET to its constituent monomers near the polymer glass transition temperature. To date, reported PET hydrolases have been sourced from a relatively narrow sequence space. Here, we identify additional PET-active biocatalysts from natural diversity by using bioinformatics and machine learning to mine 74 putative thermotolerant PET hydrolases.

Enzyme kinetics by GH7 cellobiohydrolases on chromogenic substrates is dictated by non-productive binding: insights from crystal structures and MD simulation.

Cellobiohydrolases (CBHs) in the glycoside hydrolase family 7 (GH7) (EC3.2.1.

Machine learning reveals sequence-function relationships in family 7 glycoside hydrolases.

Family 7 glycoside hydrolases (GH7) are among the principal enzymes for cellulose degradation in nature and industrially. These enzymes are often bimodular, including a catalytic domain and carbohydrate-binding module (CBM) attached via a flexible linker, and exhibit an active site that binds cello-oligomers of up to ten glucosyl moieties. GH7 cellulases consist of two major subtypes: cellobiohydrolases (CBH) and endoglucanases (EG).

Improving Enzyme Optimum Temperature Prediction with Resampling Strategies and Ensemble Learning.

Accurate prediction of the optimal catalytic temperature () of enzymes is vital in biotechnology, as enzymes with high values are desired for enhanced reaction rates. Recently, a machine learning method (temperature optima for microorganisms and enzymes, TOME) for predicting was developed. TOME was trained on a normally distributed data set with a median of 37 °C and less than 5% of values above 85 °C, limiting the method's predictive capabilities for thermostable enzymes.

Inhibition Mechanisms of 2'-Hydroxybiphenyl-2-sulfinate Desulfinase ().

Naturally occurring enzymatic pathways enable highly specific, rapid thiophenic sulfur cleavage occurring at ambient temperature and pressure, which may be harnessed for the desulfurization of petroleum-based fuel. One pathway found in bacteria is a four-step catabolic pathway (the 4S pathway) converting dibenzothiophene (DBT), a common crude oil contaminant, into 2-hydroxybiphenyl (HBP) without disrupting the carbon-carbon bonds. 2'-Hydroxybiphenyl-2-sulfinate desulfinase (), the rate-limiting enzyme in the enzyme cascade, is capable of selectively cleaving carbon-sulfur bonds.

Enabling microbial syringol conversion through structure-guided protein engineering.

Microbial conversion of aromatic compounds is an emerging and promising strategy for valorization of the plant biopolymer lignin. A critical and often rate-limiting reaction in aromatic catabolism is -aryl-demethylation of the abundant aromatic methoxy groups in lignin to form diols, which enables subsequent oxidative aromatic ring-opening. Recently, a cytochrome P450 system, GcoAB, was discovered to demethylate guaiacol (2-methoxyphenol), which can be produced from coniferyl alcohol-derived lignin, to form catechol.

Thermodynamic Signatures of Substrate Binding for Three Thermobifida fusca Cellulases with Different Modes of Action.

The enzymatic breakdown of recalcitrant polysaccharides is achieved by synergistic enzyme cocktails of glycoside hydrolases (GHs) and accessory enzymes. Many GHs are processive, meaning that they stay bound to the substrate between subsequent catalytic interactions. Cellulases are GHs that catalyze the hydrolysis of cellulose [β-1,4-linked glucose (Glc)].

Evaluation of the Immunomodulatory Effects of All-Trans Retinoic Acid Solid Lipid Nanoparticles and Human Mesenchymal Stem Cells in an A549 Epithelial Cell Line Model.

Purpose: To investigate two potential strategies aimed at targeting the inflammatory pathogenesis of COPD: a small molecule, all trans retinoic acid (atRA) and human mesenchymal stem cells (hMSCs).

Methods: atRA was formulated into solid lipid nanoparticles (SLNs) via the emulsification-ultrasonication method, and these SLNs were characterised physicochemically. Assessment of the immunomodulatory effects of atRA-SLNs on A549 cells in vitro was determined using ELISA.

Kinetic and molecular dynamics study of inhibition and transglycosylation in family 3 β-glucosidases.

β-Glucosidases enhance enzymatic biomass conversion by relieving cellobiose inhibition of endoglucanases and cellobiohydrolases. However, the susceptibility of these enzymes to inhibition and transglycosylation at high glucose or cellobiose concentrations severely limits their activity and, consequently, the overall efficiency of enzyme mixtures. We determined the impact of these two processes on the hydrolytic activity of the industrially relevant family 3 β-glucosidases from , Cel3A and Cel3B, and investigated the underlying molecular mechanisms through kinetic studies, binding free energy calculations, and molecular dynamics (MD) simulations.

Cellulose-specific Type B carbohydrate binding modules: understanding oligomeric and non-crystalline substrate recognition mechanisms.

Background: Effective enzymatic degradation of crystalline polysaccharides requires a synergistic cocktail of hydrolytic enzymes tailored to the wide-ranging degree of substrate crystallinity. To accomplish this type of targeted carbohydrate recognition, nature produces multi-modular enzymes, having at least one catalytic domain appended to one or more carbohydrate binding modules (CBMs). The Type B CBM categorization encompasses several families (i.

Hydrolysis and Transglycosylation Transition States of Glycoside Hydrolase Family 3 β-Glucosidases Differ in Charge and Puckering Conformation.

β-Glucosidases (βgls) from glycoside hydrolase family 3 play an important role in biomass degradation by catalyzing cellobiose hydrolysis. However, the hydrolysis rate decreases when the glucose product or another cellobiose competes with water to form oligosaccharides in a reaction called transglycosylation. Both reactions involve proton transfer to the acid/base residue and nucleophilic attack on the glycosyl-enzyme intermediate.

Molecular Determinants of Substrate Affinity and Enzyme Activity of a Cytochrome P450 Variant.

Cytochrome P450 catalyzes the hydroxylation and/or epoxidation of fatty acids, fatty amides, and alcohols. Protein engineering has produced P450 variants capable of accepting drug molecules normally metabolized by human P450 enzymes. The enhanced substrate promiscuity has been attributed to the greater flexibility of the lid of the substrate channel.

Desulfination by 2'-hydroxybiphenyl-2-sulfinate desulfinase proceeds electrophilic aromatic substitution by the cysteine-27 proton.

Biodesulfurization is an attractive option for enzymatically removing sulfur from the recalcitrant thiophenic derivatives that comprise the majority of organosulfur compounds remaining in hydrotreated petroleum products. Desulfurization in the bacteria follows a four-step pathway culminating in C-S bond cleavage in the 2'-hydroxybiphenyl-2-sulfinate (HBPS) intermediate to yield 2-hydroxybiphenyl and bisulfite. The reaction, catalyzed by 2'-hydroxybiphenyl-2-sulfinate desulfinase (DszB), is the rate-limiting step and also the least understood, as experimental evidence points to a mechanism unlike that of other desulfinases.

Structural and molecular dynamics studies of a C1-oxidizing lytic polysaccharide monooxygenase from Heterobasidion irregulare reveal amino acids important for substrate recognition.

Unlabelled: Lytic polysaccharide monooxygenases (LPMOs) are a group of recently discovered enzymes that play important roles in the decomposition of recalcitrant polysaccharides. Here, we report the biochemical, structural, and computational characterization of an LPMO from the white-rot fungus Heterobasidion irregulare (HiLPMO9B). This enzyme oxidizes cellulose at the C1 carbon of glycosidic linkages.

Correlation of structure, function and protein dynamics in GH7 cellobiohydrolases from , and .

Background: The ascomycete fungus is the predominant source of enzymes for industrial conversion of lignocellulose. Its glycoside hydrolase family 7 cellobiohydrolase (GH7 CBH) Cel7A constitutes nearly half of the enzyme cocktail by weight and is the major workhorse in the cellulose hydrolysis process. The orthologs from (Cel7A) and (Cel7A) show high sequence identity with Cel7A, ~ 80%, and represent naturally evolved combinations of cellulose-binding tunnel-enclosing loop motifs, which have been suggested to influence intrinsic cellobiohydrolase properties, such as endo-initiation, processivity, and off-rate.

The role of catalytic residue pK on the hydrolysis/transglycosylation partition in family 3 β-glucosidases.

β-Glucosidases (βgls) primarily catalyze the hydrolysis of the terminal glycosidic bond at the non-reducing end of β-glucosides, although glycosidic bond synthesis (called transglycosylation) can also occur in the presence of another acceptor. In the final reaction step, the glucose product or another substrate competes with water for transfer to the glycosyl-enzyme intermediate. The factors governing the balance between the two pathways are not fully known; however, the involvement of ionizable residues in binding and catalysis suggests that their pK may play a role.

Improving the thermal stability of cellobiohydrolase Cel7A from by directed evolution.

Secreted mixtures of cellulases are able to efficiently degrade cellulosic biomass to fermentable sugars at large, commercially relevant scales. Cel7A, cellobiohydrolase I, from glycoside hydrolase family 7, is the workhorse enzyme of the process. However, the thermal stability of Cel7A limits its use to processes where temperatures are no higher than 50 °C.

Inhibition of Mammalian Glycoprotein YKL-40: IDENTIFICATION OF THE PHYSIOLOGICAL LIGAND.

YKL-40 is a mammalian glycoprotein associated with progression, severity, and prognosis of chronic inflammatory diseases and a multitude of cancers. Despite this well documented association, identification of the lectin's physiological ligand and, accordingly, biological function has proven experimentally difficult. YKL-40 has been shown to bind chito-oligosaccharides; however, the production of chitin by the human body has not yet been documented.

CHARMM force field parameters for 2'-hydroxybiphenyl-2-sulfinate, 2-hydroxybiphenyl, and related analogs.

2'-Hydroxybiphenyl-2-sulfinate (HBPS) desulfinase (DszB) catalyzes the cleavage of the carbon-sulfur bond from HBPS in the final step of microbial 4S pathway desulfurization reactions. DszB is notable for its substrate specificity and exhibits product inhibition, both of which hinder the overall 4S pathway turnover rate. To understand the molecular-level contributions to substrate and inhibitor binding to DszB, we plan to perform molecular dynamic simulations bound to an array of naphthenic molecules and biphenyl analogues of HBPS.

Effect of Mutation and Substrate Binding on the Stability of Cytochrome P450BM3 Variants.

Cytochrome P450BM3 is a heme-containing enzyme from Bacillus megaterium that exhibits high monooxygenase activity and has a self-sufficient electron transfer system in the full-length enzyme. Its potential synthetic applications drive protein engineering efforts to produce variants capable of oxidizing nonnative substrates such as pharmaceuticals and aromatic pollutants. However, promiscuous P450BM3 mutants often exhibit lower stability, thereby hindering their industrial application.

Aromatic-Mediated Carbohydrate Recognition in Processive Serratia marcescens Chitinases.

Microorganisms use a host of enzymes, including processive glycoside hydrolases, to deconstruct recalcitrant polysaccharides to sugars. Processive glycoside hydrolases closely associate with polymer chains and repeatedly cleave glycosidic linkages without dissociating from the crystalline surface after each hydrolytic step; they are typically the most abundant enzymes in both natural secretomes and industrial cocktails by virtue of their significant hydrolytic potential. The ubiquity of aromatic residues lining the enzyme catalytic tunnels and clefts is a notable feature of processive glycoside hydrolases.

Plant α-glucan phosphatases SEX4 and LSF2 display different affinity for amylopectin and amylose.

The plant glucan phosphatases Starch EXcess 4 (SEX4) and Like Sex Four2 (LSF2) apply different starch binding mechanisms. SEX4 contains a carbohydrate binding module, and LSF2 has two surface binding sites (SBSs). We determined KDapp for amylopectin and amylose, and KD for β-cyclodextrin and validated binding site mutants deploying affinity gel electrophoresis (AGE) and surface plasmon resonance.