Microstructured reactors are emerging engineering tools for the development of biocatalytic conversions in continuous flow. A promising layout involves flow microchannels that are wall‐coated with enzyme. As protein immobilization within closed microstructures is challenging, we suggested a confluent design of enzyme and microreactor: fusion to the silica‐binding module Zbasic2 is used to engineer enzymes for high‐affinity oriented attachment to the plain wall surface of glass microchannels. In this study of sucrose phosphorylase, we examined the effects of multiple Zbasic2 modules in a single enzyme molecule on the activity and adsorption stability of the phosphorylase immobilized in a glass microchannel reactor. Compared to the “monovalent” enzyme, two Zbasic2 modules, present in tandem repeat at the N‐terminus, separated at the N‐ and C‐terminus of an enzyme monomer, or arranged N‐terminally in a protein homodimer, boosted the effectiveness of the immobilized phosphorylase by up to twofold. They attenuated (up to 12‐fold) the elution of the wall‐coated enzyme during continuous reactor operation. The divalent phosphorylase was distributed uniformly on the microchannel surface and approximately 70 % activity could still be retained after 690 reactor cycles. Reaction–diffusion regime analysis in terms of the second Damköhler number (DaII≤0.02) revealed the absence of mass transport limitations on the conversion rate. The synthesis of α‐d‐glucose 1‐phosphate occurred with a productivity of ∼14 mm min−1 at 50 % substrate conversion (50 mm). The use of wall‐coated enzyme microreactors in high‐performance biocatalytic reaction engineering is supported strongly.

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.

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 ∼105 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.

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D-Apiose is a C-branched pentose sugar important for plant cell wall development. Its biosynthesis as UDP-D-apiose involves decarboxylation of the UDP-D-glucuronic acid precursor coupled to pyranosyl-to-furanosyl sugar ring contraction. This unusual multistep reaction is catalyzed within a single active site by UDP-D-apiose/UDP-D-xylose synthase (UAXS). Here, we decipher the UAXS catalytic mechanism based on crystal structures of the enzyme from Arabidopsis thaliana, molecular dynamics simulations expanded by QM/MM calculations, and mutational-mechanistic analyses. Our studies show how UAXS uniquely integrates a classical catalytic cycle of oxidation and reduction by a tightly bound nicotinamide coenzyme with retro-Users may view, print, copy, and download text and data-mine the content in such documents, for the purposes of academic research, subject always to the full Conditions of use:

Biosynthesis of 6-deoxy sugars, including l -fucose, involves a mechanistically complex, enzymatic 4,6-dehydration of hexose nucleotide precursors as the first committed step. Here, we determined pre- and postcatalytic complex structures of the human GDP-mannose 4,6-dehydratase at atomic resolution. These structures together with results of molecular dynamics simulation and biochemical characterization of wildtype and mutant enzymes reveal elusive mechanistic details of water elimination from GDP-mannose C5″ and C6″, coupled to NADP-mediated hydride transfer from C4″ to C6″. We show that concerted acid–base catalysis from only two active-site groups, Tyr 179 and Glu 157 , promotes a syn 1,4-elimination from an enol (not an enolate) intermediate. We also show that the overall multistep catalytic reaction involves the fewest position changes of enzyme and substrate groups and that it proceeds under conserved exploitation of the basic (minimal) catalytic machinery of short-chain dehydrogenase/reductases.

The Cori ester ␣-D-glucose 1-phosphate (␣Glc 1-P) is a high-energy intermediate of cellular carbohydrate metabolism. Its glycosidic phosphomonoester moiety primes ␣Glc 1-P for flexible exploitation in glucosyl and phosphoryl transfer reactions. Two structurally and mechanistically distinct sugar-phosphate phosphatases from Escherichia coli were characterized in this study for utilization of ␣Glc 1-P as a phosphoryl donor substrate. The agp gene encodes a periplasmic ␣Glc 1-P phosphatase (Agp) belonging to the histidine acid phosphatase family. Had13 is from the haloacid dehydrogenase-like phosphatase family. Cytoplasmic expression of Agp (in E. coli Origami B) gave a functional enzyme preparation (k cat for phosphoryl transfer from ␣Glc 1-P to water, 40 s ؊1 ) that was shown by mass spectrometry to exhibit no free cysteines and the native intramolecular disulfide bond between Cys 189 and Cys 195 . Enzymatic phosphoryl transfer from ␣Glc 1-P to water in H 2 18 O solvent proceeded with complete 18 O label incorporation into the phosphate released, consistent with catalytic reaction through O-1-P, but not C-1-O, bond cleavage. Hydrolase activity of both enzymes was not restricted to a glycosidic phosphomonoester substrate, and D-glucose 6-phosphate was converted with a k cat similar to that of ␣Glc 1-P. By examining phosphoryl transfer from ␣Glc 1-P to an acceptor substrate other than water (D-fructose or D-glucose), we discovered that Agp exhibited pronounced synthetic activity, unlike Had13, which utilized ␣Glc 1-P mainly for phosphoryl transfer to water. By applying D-fructose in 10-fold molar excess over ␣Glc 1-P (20 mM), enzymatic conversion furnished D-fructose 1-phosphate as the main product in a 55% overall yield. Agp is a promising biocatalyst for use in transphosphorylation from ␣Glc 1-P. Phosphorylation of sugar substrates is a common biochemical transformation of central importance to cellular metabolism (1-3). It usually involves phosphoryl transfer from a phosphoactivated donor substrate, such as ATP, to an acceptor group, typically a hydroxyl, on the sugar backbone (4-7). Various phosphotransferases (EC 2.7) catalyze sugar phosphorylation (8-12). In an alternative reaction catalyzed by glycoside phosphorylases (EC 2.4), where phosphorylation occurs exclusively at the sugar's anomeric position, a glycosyl residue is transferred from a sugar donor substrate to phosphate (Fig. 1A) (13, 14). The phosphomonoester moiety attached to sugars is a key element of biological recognition, across all steps of glycolysis, for example, and it serves to prime sugars for further conversion in different biochemical pathways (15-18). It is known from intracellular-metabolite-profiling studies that changes in concentrations of common sugar phosphates (e.g., D-glucose 6-phosphate [Glc 6-P] and D-fructose 6-phosphate [Fru 6-P]) are often linked to major alterations in cellular physiology (19)(20)(21)(22). Due to the requirement for authentic reference material in different biological investigations, there is considerable ...