C-Analogues of the canonical N-nucleosides have considerable importance in medicinal chemistry and are promising building blocks of xenobiotic nucleic acids (XNA) in synthetic biology. Although well ...established for synthesis of N-nucleosides, biocatalytic methods are lacking in C-nucleoside synthetic chemistry. Here, we identify pseudouridine monophosphate C-glycosidase for selective 5-β-C-glycosylation of uracil and derivatives thereof from pentose 5-phosphate (D-ribose, 2-deoxy-D-ribose, D-arabinose, D-xylose) substrates. Substrate requirements of the enzymatic reaction are consistent with a Mannich-like addition between the pyrimidine nucleobase and the iminium intermediate of enzyme (Lys166) and open-chain pentose 5-phosphate. β-Elimination of the lysine and stereoselective ring closure give the product. We demonstrate phosphorylation-glycosylation cascade reactions for efficient, one-pot synthesis of C-nucleoside phosphates (yield: 33 - 94%) from unprotected sugar and nucleobase. We show incorporation of the enzymatically synthesized C-nucleotide triphosphates into nucleic acids by RNA polymerase. Collectively, these findings implement biocatalytic methodology for C-nucleotide synthesis which can facilitate XNA engineering for synthetic biology applications.
Cellulose-based materials are produced industrially in countless varieties via top-down processing of natural lignocellulose substrates. By contrast, cellulosic materials are only rarely prepared via ...bottom up synthesis and oligomerization-induced self-assembly of cellulose chains. Building up a cellulose chain via precision polymerization is promising, however, for it offers tunability and control of the final chemical structure. Synthetic cellulose derivatives with programmable material properties might thus be obtained. Cellodextrin phosphorylase (CdP; EC 2.4.1.49) catalyzes iterative β-1,4-glycosylation from α-d-glucose 1-phosphate, with the ability to elongate a diversity of acceptor substrates, including cellobiose, d-glucose and a range of synthetic glycosides having non-sugar aglycons. Depending on the reaction conditions leading to different degrees of polymerization (DP), short-chain soluble cello-oligosaccharides (COS) or insoluble cellulosic materials are formed. Here, we review the characteristics of CdP as bio-catalyst for synthetic applications and show advances in the enzymatic production of COS and reducing end-modified, tailored cellulose materials. Recent studies reveal COS as interesting dietary fibers that could provide a selective prebiotic effect. The bottom-up synthesized celluloses involve chains of DP ≥ 9, as precipitated in solution, and they form ~5 nm thick sheet-like crystalline structures of cellulose allomorph II. Solvent conditions and aglycon structures can direct the cellulose chain self-assembly towards a range of material architectures, including hierarchically organized networks of nanoribbons, or nanorods as well as distorted nanosheets. Composite materials are also formed. The resulting materials can be useful as property-tunable hydrogels and feature site-specific introduction of functional and chemically reactive groups. Therefore, COS and cellulose obtained via bottom-up synthesis can expand cellulose applications towards product classes that are difficult to access via top-down processing of natural materials.
•Cellodextrin phosphorylases (EC 2.4.1.49) as biocatalyst for cello-oligomer synthesis•Structure, specificity and catalytic properties of cellodextrin phosphorylase•Synthesis of reducing end-modified cellulose oligomers for property-tunable materials•Synthesis of soluble cello-oligosaccharides by cascades of glycoside phosphorylases•Applied potential of products synthesized by cellodextrin phosphorylase
Abstract
Enzyme-catalyzed iterative
β
-1,4-glycosylation of
β
-glycosides is promising for bottom-up polymerization of reducing-end-modified cello-oligosaccharide chains. Self-assembly of the chains ...from solution yields crystalline nanocellulose materials with properties that are tunable by the glycoside group used. Cellulose chains with a reducing-end thiol group are of interest to install a controllable pattern of site-selective modifications into the nanocellulose material. Selection of the polymerizing enzyme (cellodextrin phosphorylase; CdP) was pursued here to enhance the synthetic precision of
β
-1-thio-glucose conversion to generate pure “1-thio-cellulose” (≥95%) unencumbered by plain (unlabeled) cellulose resulting from enzymatic side reactions. The CdP from
Clostridium stercorarium
(
Cs
CdP) was 21 times more active on
β
-1-thio-glucose (0.17 U/mg; 45 °C) than the CdP from
Clostridium cellulosi
(
Cc
CdP), and it lacked hydrolase activity, which is substantial in
Cc
CdP, against the α-
d
-glucose 1-phosphate donor substrate. The combination of these enzyme properties indicated that
Cs
CdP is a practical catalyst for 1-thio-cellulose synthesis directly from
β
-1-thio-glucose (8 h; 25 mol% yield) that does not require a second enzyme (cellobiose phosphorylase), which was essential when using the less selective
Cc
CdP. The 1-thio-cellulose chains had an average degree of polymerization of ∼10 and were assembled into highly crystalline cellulose II crystallinity material.
Because of their efficiency, selectivity, and environmental sustainability, there are significant opportunities for enzymes in chemical synthesis and biotechnology. However, as the three-dimensional ...active structure of enzymes is predominantly maintained by weaker noncovalent interactions, thermal, pH, and chemical stressors can modify or eliminate activity. Metal–organic frameworks (MOFs), which are extended porous network materials assembled by a bottom-up building block approach from metal-based nodes and organic linkers, can be used to afford protection to enzymes. The self-assembled structures of MOFs can be used to encase an enzyme in a process called encapsulation when the MOF is synthesized in the presence of the biomolecule. Alternatively, enzymes can be infiltrated into mesoporous MOF structures or surface bound via covalent or noncovalent processes. Integration of MOF materials and enzymes in this way affords protection and allows the enzyme to maintain activity in challenge conditions (e.g., denaturing agents, elevated temperature, non-native pH, and organic solvents). In addition to forming simple enzyme/MOF biocomposites, other materials can be introduced to the composites to improve recovery or facilitate advanced applications in sensing and fuel cell technology. This review canvasses enzyme protection via encapsulation, pore infiltration, and surface adsorption and summarizes strategies to form multicomponent composites. Also, given that enzyme/MOF biocomposites straddle materials chemistry and enzymology, this review provides an assessment of the characterization methodologies used for MOF-immobilized enzymes and identifies some key parameters to facilitate development of the field.
•Advanced characterization of immobilized enzymes is important for heterogeneous biocatalyst development.•Structural and in-operando studies contribute to identification of factors governing the ...activity of immobilized enzymes.•Protein visualization in solid supports is used to facilitate enzyme loading in high quantity and quality.•Elucidation of structural features of immobilized enzymes on solid support remains challenging.•In-operando opto-chemical internal sensing is prominently used to characterize the biocatalyst's internal environment.
Like in chemical catalysis, there is a clear trend in biocatalysis to carry out synthetic transformations at the manufacturing scale heterogeneously catalyzed. Recycling of insoluble catalysts is simplified, and continuous reactor development thus promoted. Heterogeneous biocatalysis usually involves enzymes immobilized on mesoporous solid supports that offer a large internal surface area. Unraveling enzyme behavior under the confinement of a solid surface and its effect on the catalytic reaction in heterogeneous environment present longstanding core problems of biocatalysis with immobilized enzymes. Progress in deepening the mechanistic understanding of heterogeneous biocatalytic conversions is often restrained by severe limitations in methodology applicable to a direct characterization of solid-supported enzymes. Here we highlight recent evidence from the analysis of protein distribution on porous solid support using microscopic imaging methods with spatiotemporal resolution capability. We also show advance in the use of spectroscopic methods for the analysis of protein conformation on solid support. Methods of direct characterization of activity and stability of immobilized enzymes as heterogeneous biocatalysts are described and their important roles in promoting rational biocatalyst design as well as optimization and control of heterogeneously catalyzed processes are emphasized.
The liquid milieu in which enzymes operate when they are immobilized in solid materials can be quite different from the milieu in bulk solution. Important differences are in the substrate and product ...concentration but also in pH and ionic strength. The internal milieu for immobilized enzymes is affected by the chemical properties of the solid material and by the interplay of reaction and diffusion. Enzyme performance is influenced by the internal milieu in terms of catalytic rate ("activity") and stability. Elucidation, through direct measurement of differences in the internal as compared to the bulk milieu is, therefore, fundamentally important in the mechanistic characterization of immobilized enzymes. The deepened understanding thus acquired is critical for the rational development of immobilized enzyme preparations with optimized properties. Herein we review approaches by opto-chemical sensing to determine the internal milieu of enzymes immobilized in porous particles. We describe analytical principles applied to immobilized enzymes and focus on the determination of pH and the O
concentration. We show measurements of pH and O
with spatiotemporal resolution, using in operando analysis for immobilized preparations of industrially important enzymes. The effect of concentration gradients between solid particle and liquid bulk on enzyme performance is made evident and quantified. Besides its use in enzyme characterization, the method can be applied to the development of process control strategies.
As a crucial factor of their therapeutic efficacy, the currently marketed mRNA vaccines feature uniform substitution of uridine (U) by the corresponding C-nucleoside, pseudouridine (Ψ), in ...1-N-methylated form. Synthetic supply of the mRNA building block (1-N-Me-Ψ-5'-triphosphate) involves expedient access to Ψ as the principal challenge. Here, we show selective and atom-economic 1N-5C rearrangement of β-D-ribosyl on uracil to obtain Ψ from unprotected U in quantitative yield. One-pot cascade transformation of U in four enzyme-catalyzed steps, via D-ribose (Rib)-1-phosphate, Rib-5-phosphate (Rib5P) and Ψ-5'-phosphate (ΨMP), gives Ψ. Coordinated function of the coupled enzymes in the overall rearrangement necessitates specific release of phosphate from the ΨMP, but not from the intermediary ribose phosphates. Discovery of Yjjg as ΨMP-specific phosphatase enables internally controlled regeneration of phosphate as catalytic reagent. With driving force provided from the net N-C rearrangement, the optimized U reaction yields a supersaturated product solution (∼250 g/L) from which the pure Ψ crystallizes (90% recovery). Scale up to 25 g isolated product at enzyme turnovers of ∼10
mol/mol demonstrates a robust process technology, promising for Ψ production. Our study identifies a multistep rearrangement reaction, realized by cascade biocatalysis, for C-nucleoside synthesis in high efficiency.
Understanding enzyme catalysis as connected to protein motions is a major challenge. Here, based on temperature kinetic studies combined with isotope effect measurements, we obtain energetic ...description of C-H activation in NAD-dependent UDP-glucuronic acid C4 epimerase. Approach from the ensemble-averaged ground state (GS) to the transition state-like reactive conformation (TSRC) involves, alongside uptake of heat (
= 54 kJ mol
), significant loss in entropy (
= 20 kJ mol
; 298 K) and negative activation heat capacity (
= -0.64 kJ mol
K
). Thermodynamic changes suggest the requirement for restricting configurational freedom at the GS to populate the TSRC. Enzyme variants affecting the electrostatic GS preorganization reveal active-site interactions important for precise TSRC sampling and H-transfer. Collectively, our study captures thermodynamic effects associated with TSRC sampling and establishes rigid positioning for C-H activation in an enzyme active site that requires conformational flexibility in fulfillment of its natural epimerase function.
Mechanistic implications: Distinct active‐site motifs in plant aryl glucosyltransferases of the GT‐1 family differentiate between C‐ and O‐glycosylation activity on a phloretin acceptor. In the ...implicated protein design principle the exchange of active‐site motifs results in reversible switch between C/O‐glycoside specificity. The proposed mechanism of the C‐glycosyltransferase involves direct nucleophilic displacement at the anomeric carbon.
Lytic polysaccharide monooxygenase (LPMO) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. Used in combination with hydrolytic enzymes, LPMO appears ...to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. LPMO activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose surface is lacking. Specificity of LPMO for degrading ordered crystalline and unordered amorphous cellulose material of the substrate surface is also unknown. We show by fluorescence dye adsorption analyzed with confocal laser scanning microscopy that a LPMO (from Neurospora crassa) introduces carboxyl groups primarily in surface-exposed crystalline areas of the cellulosic substrate. Using time-resolved in situ atomic force microscopy we further demonstrate that cellulose nano-fibrils exposed on the surface are degraded into shorter and thinner insoluble fragments. Also using atomic force microscopy, we show that prior action of LPMO enables cellulases to attack otherwise highly resistant crystalline substrate areas and that it promotes an overall faster and more complete surface degradation. Overall, this study reveals key characteristics of LPMO action on the cellulose surface and suggests the effects of substrate morphology on the synergy between LPMO and hydrolytic enzymes in cellulose depolymerization.
Lytic polysaccharide monooxygenase (LPMO) has recently been discovered to depolymerize cellulose.
Dynamic imaging was applied to reveal the effects of LPMO and cellulase activity on solid cellulose surface.
Critical features of surface morphology for LPMO synergy with cellulases are recognized.
Direct insights into cellulose deconstruction by LPMO alone and in synergy with cellulases are obtained.
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