Mechanism and biomass association of glucuronoyl esterase: an α/β hydrolase with potential in biomass conversion

Abstract

Glucuronoyl esterases (GEs) are α/β serine hydrolases and a relatively new addition in the toolbox to reduce the recalcitrance of lignocellulose, the biggest obstacle in cost-effective utilization of this important renewable resource. While biochemical and structural characterization of GEs have progressed greatly recently, there have yet been no mechanistic studies shedding light onto the rate-limiting steps relevant for biomass conversion. The bacterial GE OtCE15A possesses a classical yet distinctive catalytic machinery, with easily identifiable catalytic Ser/His completed by two acidic residues (Glu and Asp) rather than one as in the classical triad, and an Arg side chain participating in the oxyanion hole. By QM/MM calculations, we identified deacylation as the decisive step in catalysis, and quantified the role of Asp, Glu and Arg, showing the latter to be particularly important. The results agree well with experimental and structural data. We further calculated the free-energy barrier of post-catalysis dissociation from a complex natural substrate, suggesting that in industrial settings non-catalytic processes may constitute the rate-limiting step, and pointing to future directions for enzyme engineering in biomass utilization.

Glucuronoyl esterases – enzymes to decouple lignin and carbohydrates and enable better utilization of renewable plant biomass

“…As already shown by initial crystallographic ligand binding studies ( Figure 4 ) [ 43 ], the GlcA moiety of the esters cleaved by GEs binds in a pocket located above the main parallel β-sheet of the ABH fold ( Figure 3 ), which is located on a fairly open surface in the structurally characterized fungal members of CE15 [ 38 , 43 , 44 ]. In structurally characterized bacterial enzymes, sequence insertions create high ridges on one or two sides of the substrate binding surface ( Figure 3 ), indicating that the enzymes might interact more intimately with their complex substrate than their fungal counterparts [ 28 , 31 , 39 , 42 , 45 , 46 ].…”

“…In addition to the aforementioned product complexes, covalent intermediates have been trapped in crystals with catalytically impaired versions of the same enzymes, where either the catalytic His or a conserved active site Arg had been mutated to Ala for Ot CE15A [ 42 , 46 ], or using the wild-type Cu GE [ 38 ]. The highly conserved Arg in the catalytic cleft is important for stabilization of the oxyanion hole during catalysis, a function which in many ABHs is fulfilled by main chain nitrogen atoms.…”

“…Other ‘inactive’ GEs have been reported (with different sequence features), which may suggest yet undiscovered natural substrates in the family [ 47 , 48 ]. The accumulated structural information has been the basis for the first computational investigations of the catalytic mechanism of the model GE Ot CE15A using QM/MM [ 46 ]. The study confirmed that for this enzyme, the deacylation step is rate-limiting, though only by a small margin.…”

Molecular mechanism of LIP05 derived from Monascus purpureus YJX-8 for synthesizing fatty acid ethyl esters under aqueous phase

“…LIP05 had two domains and belonged to α/β fold hydrolase, the core catalytic domain consisted of 5 β-sheets [β1 (Tyr66-Ala71), β2 (Leu101-Gly105), β3 (His143-Tyr149), β4 (Ser173-Phe177), and β5 (Val194-Ala197)] surrounded by 7 α-helices [α2 (Gly85-Val96), α3 (Ile113-Ala118), α4 (Asp121-Thr138), α5 (Gln151-Lys162), α6 (Ala165-Trp170), α7 (Asn203-Gln206), and α8 (Pro213-Ala232)], the lid domain consisted of 1 α-helix [α1 (Ile30-Thr48); Figures 1A , B ]. The catalytic triad was Ser150-His215-Asp202 ( Supplementary Figure S1 ), and belonged to the classical catalytic triad of α/β fold hydrolase ( Rauwerdink and Kazlauskas, 2015 ; Zong et al, 2022 ). Ser150 was located on the “nucleophile elbow” connecting β3 and α5, His215 was located on the α8, and Asp202 was located on the loop between β5 and α7 ( Figures 1A , B ).…”

Covalent‐Bonding Oxidation Group and Titanium Cluster to Synthesize a Porous Crystalline Catalyst for Selective Photo‐Oxidation Biomass Valorization

“…[1] The efficient valorization of biomass could play a role in solving energy and environmental problems for future sustainable development. [2] For example, 5-hydroxymethylfurfural (HMF), the dehydration product of C6 carbohydrates, is a vital platform chemical for biomass-derived intermediates, which has been utilized for the syntheses of a series of pharmaceuticals, monomers and fine chemicals (e.g., maleic anhydride (MA), 2,5-diformylfuran (DFF), 5-hydroxymethyl-2-furancarboxylic acid (HMFCA), and 2,5-furandicarboxylic acid (FDCA), etc.). [3] Among them, FDCA is identified by the department of energy (U.S.A) to be a key bio-derived chemical and most importantly, a vital monomer for the synthesis of bio-based polyethylene furandicarboxylate (PEF) that has much potential to replace commonly applied polyethylene terephthalate (PET).…”