Structure‐Based Enhancement of the First Anomeric Glucokinase

Abstract

Many pharmaceutically important compounds derive from carbohydrate-containing natural products, and sugar moieties of these molecules have been proven to play an important role in drug targeting, biological activity, and pharmacology. [1±7] Thus, altering the glycosylation of natural products would significantly contribute to the diversity of novel therapeutics. Among a number of routes for altering glycosylation, naturalproduct glycorandomization is one of the most efficient approaches for complex secondary metabolites.[8±14] In vitro glycorandomization (IVG) technology establishes a promiscuous chemoenzymatic system to quickly build diverse glycorandomized libraries based on natural-product structures (Figure 1 a). This process utilizes chemical synthesis to provide a repertoire of unique sugar precursors, and three promiscuous enzymes to activate (anomeric sugar kinases and nucleotidylyltransferases) and attach (glycosyltransferases) these carbohydrate libraries to various complex natural-product aglycons. Anomeric sugar kinases, as key components of glycorandomization, directly determine the availability of sugar phosphates for chemoenzymatic routes toward complex glycoconjugates. Thus, generation of a flexible sugar kinase capable of accepting a wide array of monosaccharide substrates would directly enhance the efficiency of IVG. Of particular interest are d-glucoconfigured scaffolds given their prevalence in glycosylated natural products (Figure 1 b), [1,3,4,6] glycoproteins, [15±18] as well as many bacterial and eukaryotic cell-surface glycosylation patterns.[18±20]To date, an enzyme capable of d-glucose (Figure 2, 13) anomeric phosphorylation (a glucokinase, or GlcK) remains elusive. Toward this goal, directed evolution of E. coli galactokinase (GalK) led to the Y371H variant with remarkably widened substrate flexibility at C-2, C-3, and C-5 of the sugar. Yet, all sugars tested containing alternative C-4 substitutions (e.g. 4-deoxy-d-galactose, 12 or 13, Figure 2) were not accepted as substrates for the evolved catalyst.[21] As an alternative approach, the recently elucidated structure of Lactococcus lactis GalK provides a template for rational sugar kinase engineering.[22] The L. lactis GalK structure reveals that the strongly conserved active-site residues Asp45 and Tyr233 hydrogen bond with the critical galactose C-4 axial hydroxyl.[22] While a recent saturation mutagenesis of the equivalent residues within the E. coli GalK revealed the role of the active-site tyrosine to be hydrophobic stacking and expanded the substrate range to include 12, [23] a GlcK has still not been found. To continue our quest for an anomeric GlcK and further explore the relevance of the L. lactis structural model to other GalKs, we report the generation and characterization of the Y385H L. lactis GalK mutant-the promiscuous E. coli equivalent of which was discovered through directed evolution.[21] Remarkably, we reveal that this L. lactis variant displays a substantial degree of kinase activity toward several C-4-substitut...

Characterization of Meiothermus taiwanensis Galactokinase and its Use in the One‐Pot Enzymatic Synthesis of Uridine Diphosphate‐Galactose and the Chemoenzymatic Synthesis of the Carbohydrate Antigen Stage Specific Embryonic Antigen‐3

“…In the preliminary enzymatic activity screening assay, the reaction of MtGalK was performed in a solution (100-200 mL) containing 8 mM Gal, 10 mM ATP, 5 mM MgCl 2 , 50 mM HEPES (pH 7.5), and 1 mg mL À1 purified MtGalK. The reaction was incubated at 37 8C for 0, 1, 2, 3, 4,5,6,7,8,9,10,15,20,25,30,40,50, 60, 90 and 120 min and was quenched by the addition of 200 mM EDTA to a final EDTA concentration of 20 mM. In a further optimal reaction condition analysis (temperature, metal ion cofactor and pH), the reaction was incubated for 5 min then quenched by the addition of EDTA.…”

The Molecular Architecture of Human N-Acetylgalactosamine Kinase

“…Thus far, a variant of the E. coli galactokinase (Y371H) has demonstrated a marked increase in substrate flexibility with respect to modifications at C-2, C-3, and C-5 but not at C-4 (38). Quite strikingly, the same mutation in the L. lactis enzyme (Y385H) resulted in a protein displaying kinase activity toward several C-4-substituted sugars (39). These contrasting results from the E. coli and L. lactis enzymes emphasize the advantages of optimizing additional enzymes for synthetic purposes.…”

A comparison of sugar indicators enables a universal high-throughput sugar-1-phosphate nucleotidyltransferase assay

“…The prior successful applications of the DNS reducing sugar assay for kinetic analysis and substrate specificity determinations in the context of sugar kinase engineering and directed evolution inspired the investigation of a similar strategy for nucleotidyltransferase activity assays [32][33][34][35][36]. Specifically, we hypothesized that the addition of sufficient levels of phosphatase to the nucleotidyltransferase reaction mixture at any given time point would rapidly hydrolyze remaining sugar-1-phosphate to provide free reducing sugar, a substance directly detectable with available free sugar assays (Fig.…”

“…We previously described the development and application of a robust, high-throughput colorimetric 'DNS assay' for the kinetic characterization, directed-evolution and structurebased engineering promiscuous anomeric kinases [32][33][34][35][36]. This assay strategy, based upon an oxidation-reduction reaction between dinitrosalicylic acid (DNS, the oxidant) and a reducing sugar, was readily applicable to a wide range of free sugars in the context of crude lysates and presented a general assay platform potentially amenable to a variety of sugar-processing enzymes.…”