Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Williams, Gavin J.; Domann, Silvie; Nelson, Adam; Berry, Alan

doi:10.1073/pnas.0635924100

Cited by 110 publications

(78 citation statements)

References 33 publications

(45 reference statements)

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“…Activity was assessed using a standard enzyme-coupled assay linking cleavage of the phosphorylated sugar to oxidation of ␤-NADH by glycerol-3-phosphate dehydrogenase, which was measured spectroscopically. This analysis revealed that LacD.2 readily cleaved both TBP and FBP (Table 1) at rates similar to those reported previously (12) and similar to those with the class II TBP aldolase found in E. coli (22). When the S. pyogenes enzymes were compared, it was found that the k cat of LacD.1 was similar to that of LacD.2, although it was slightly lower for both substrates (approximately 1.5-to 2-fold) ( Table 1).…”

Section: Resultssupporting

confidence: 71%

Adaptive Evolution of the Streptococcus pyogenes Regulatory Aldolase LacD.1

Cusumano¹,

Caparon²

2013

Journal of Bacteriology

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In the human-pathogenic bacterium Streptococcus pyogenes, the tagatose bisphosphate aldolase LacD.1 likely originated through a gene duplication event and was adapted to a role as a metabolic sensor for regulation of virulence gene transcription. Although LacD.1 retains enzymatic activity, its ancestral metabolic function resides in the LacD.2 aldolase, which is required for the catabolism of galactose. In this study, we compared these paralogous proteins to identify characteristics correlated with divergence and novel function. Surprisingly, despite the fact that these proteins have identical active sites and 82% similarity in amino acid sequence, LacD.1 was less efficient at cleaving both fructose and tagatose bisphosphates. Analysis of kinetic properties revealed that LacD.1's adaptation was associated with a decrease in k cat and an increase in K m . Construction and analysis of enzyme chimeras indicated that non-active-site residues previously associated with the variable activities of human aldolase isoenzymes modulated LacD.1's affinity for substrate. Mutant LacD.1 proteins engineered to have LacD.2-like levels of enzymatic efficiency lost the ability to function as regulators, suggesting that an alteration in efficiency was required for adaptation. In competition under growth conditions that mimic a deep-tissue environment, LacD.1 conferred a significant gain in fitness that was associated with its regulatory activity. Taken together, these data suggest that LacD.1's adaptation represents a form of neofunctionalization in which duplication facilitated the gain of regulatory function important for growth in tissue and pathogenesis.

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Section: Resultssupporting

confidence: 71%

Adaptive Evolution of the Streptococcus pyogenes Regulatory Aldolase LacD.1

Cusumano¹,

Caparon²

2013

Journal of Bacteriology

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“…The alternative to rational engineering is enzyme evolution, a process primarily dependent on the availability of a selection or high-throughput screen for the desired enhanced or altered enzymatic properties (22)(23)(24). With respect to carbohydrate enzymology, recent applications include the tagatose-1,6-bisphosphate aldolase modified by in vitro evolution toward an unnatural stereoselectivity (25), an evolved N-acetylneuraminic acid aldolase for L-sialic acid synthesis (26), or a 2-deoxy-D-ribose-5-phosphate aldolase with an expanded substrate range after site-directed mutagenesis (27). Usually, in vitro evolution strategies include error-prone PCR for gene diversification (28,29) and͞or locating critical amino acid residues for saturation mutagenesis (30) or, more prominently, shuffling of fragmented diversified genes or gene families according to a number of different protocols (31)(32)(33)(34)(35)(36).…”

mentioning

confidence: 99%

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Hoffmeister

Yang²,

Liu³

et al. 2003

Proceedings of the National Academy of Sciences

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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 ...

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“…The assay was used by Williams et al . [14] and Woodhall et al . [15] for evolving sialic acid aldolases to accept non -natural aldehyde acceptors.…”

Section: Transaldolases and Transketolasesmentioning

confidence: 99%

Fluorescence Assays for Biotransformations

Reymond

2008

Modern Biocatalysis

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Modifying the stereochemistry of an enzyme-catalyzed reaction by directed evolution

Cited by 110 publications

References 33 publications

Adaptive Evolution of the Streptococcus pyogenes Regulatory Aldolase LacD.1

Adaptive Evolution of the Streptococcus pyogenes Regulatory Aldolase LacD.1

Creation of the first anomeric D/L-sugar kinase by means of directed evolution

Fluorescence Assays for Biotransformations

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