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 ...

Natural‐Product Sugar Biosynthesis and Enzymatic Glycodiversification

“…To facilitate the preparation of NDP-sugars, directed evolution and structurebased protein engineering have been used to create sugar biosynthetic enzymes with broader substrate specificity. For example, a single round of random mutagenesis on the galactokinase (galK) gene from E. coli [287] was sufficient to generate a GalK variant (Y371 H) that tolerates substitutions at C-2, C-3, C-5, and C-6 of d-galactose, but which maintains a stringent requirement for the axial 4-OH group. This mutant can also phosphorylate two l-sugars (278 and 279, Scheme 24).…”

Directed evolution: tailoring biocatalysts for industrial applications

“…Examples include ADP-glucose pyrophosphorylase (Salamone et al, 2002), amylosucrase (van der Veen et al, 2004), cytochrome P450 (Mandai et al 2009), lipase (Shu et al, 2010), aldolase (Williams et al, 2003), sugar kinase (Hoffmeister et al, 2003), cellulase (Kim et al, 2000), amylases (Bessler et al, 2003), xylanases (Mchunu et al, 2009; Critical Reviews in Biotechnology Downloaded from informahealthcare.com by Serials Unit -Library on 10/22/12…”

Pathway and Enzyme Engineering and Applications for Glycodiversification

“…One striking example is the work of Hoffmeister and Thorson [170]. Utilizing a high-throughput colorimetric screen, one particular GalK mutant carrying a single amino acid exchange (Y371H) was obtained in a single round of random mutagenesis on the galactokinase (galK ) gene from E. coli .…”