Creation of the first anomeric d / l -sugar kinase by means of directed evolution

Abstract

Chemoenzymatic routes toward complex glycoconjugates often depend on the availability of sugar-1-phosphates. Yet the chemical synthesis of these vital components is often tedious, whereas natural enzymes capable of anomeric phosphorylation are known to be specific for one or only a few monosaccharides. Herein we describe the application of directed evolution and a high-throughput multisugar colorimetric screen to enhance the catalytic capabilities of the Escherichia coli galactokinase GalK. From this approach, one particular GalK mutant carrying a single amino acid exchange (Y371H) displayed a surprisingly substantial degree of kinase activity toward sugars as diverse as D-galacturonic acid, D-talose, L-altrose, and L-glucose, all of which failed as wild-type GalK substrates. Furthermore, this mutant provides enhanced turnover of the small pool of sugars converted by the wild-type enzyme. Comparison of this mutation to the recently solved structure of Lactococcus lactis GalK begins to provide a blueprint for further engineering of this vital class of enzyme. In addition, the rapid access to such promiscuous sugar C-1 kinases will significantly enhance accessibility to natural and unnatural sugar-1-phosphates and thereby impact both in vitro and in vivo glycosylation methodologies, such as natural product glycorandomization.galactokinase ͉ glycorandomization ͉ in vitro evolution ͉ enzyme M any clinically important medicines are derived from glycosylated natural products, the D-or L-sugar substituents of which often dictate their overall biological activity. This paradigm is found throughout the anticancer and antiinfective arenas with representative clinical examples (Fig. 1a), including enediynes (calicheamicin, 1), polyketides (doxorubicin, 2; erythromycin, 3), indolocarbazoles (staurosporine, 4), nonribosomal peptides (vancomycin, 5), polyenes (nystatin, 6), coumarins (novobiocin, 7), and cardiac glycosides (digitoxin, 8) (1-7). Given the importance of the sugars attached to these and other biologically significant metabolites, extensive effort has been directed in recent years toward altering sugars as a means to enhance or alter natural product-based therapeutics by both in vivo and in vitro approaches (ref. 8 and references therein). Among these, in vitro glycorandomization (IVG) makes use of the inherent or engineered substrate promiscuity of nucleotidylyltransferases and glycosyltransferases to activate and attach chemically synthesized sugar precursors to various natural product scaffolds (6,7,(9)(10)(11)(12)(13)(14)(15), the advantage of which is the ability to efficiently incorporate highly functionalized ''unnatural'' sugar substitutions into the corresponding natural product scaffold (Fig. 1b). In a recent demonstration of IVG, Ͼ50 analogs of 5 were generated, some of which displayed enhanced and distinct antibacterial profiles from the parent natural product (13).As starting materials, sugar phosphates play a key role in the entire IVG process. Thus, the ability to rapidly construct sugar phosphate ...

Expanding the Nucleotide and Sugar 1-Phosphate Promiscuity of Nucleotidyltransferase RmlA via Directed Evolution

“…The process has also been specifically applied to two enzymes relevant to glycosylation, including those utilized for glycorandomization (32)(33)(34)(35). To date, the main limitation for sugar-1-phosphate nucleotidyltransferase directed evolution has been a lack of a facile high-throughput screen.…”

Expression, purification, crystallization and preliminary X-ray diffraction analysis of galactokinase fromPyrococcus horikoshii

“…In humans, deficiencies of this enzyme can result in type II galactosaemia (MIM 230200), the main symptom of which is cataract development (Holton et al, 2001). Recently, Escherichia coli galactokinase has been utilized via a directed-evolution approach to create natural and unnatural sugar 1-phosphates in a process of glycorandomization, with the aim of enhancement of drug-discovery efforts (Hoffmeister et al, 2003;Yang et al, 2005).…”

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.…”