The impact of enzyme engineering upon natural product glycodiversification

Williams, Gavin J.; Gantt, Richard W.; Thorson, Jon S.

doi:10.1016/j.cbpa.2008.07.013

Cited by 90 publications

(75 citation statements)

References 72 publications

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“…Specifically, this study focused upon two series of combinations: (i) improving the ATP/CTP activities of the "NTP generalist" (substituted at Gln 91 ) via combining with advantageous Gln 27 mutations and (ii) improving targeted sugar 1-phosphate activities through combining the most productive mutations at Gln 27 , Leu 89 , Tyr 146 , and Trp 224 . In most cases, the combination of two mutations enhanced activity as expected (Figs.…”

Section: Table 2 Specific Activity Of Selected Sugar Mutantsmentioning

confidence: 99%

“…One of the most permissive and best studied nucleotidyltransferases to date is the glucose-1-phosphate thymidylyltransferase from Salmonella enterica typhimurium LT2 (RmlA; Fig. 1B) (24,27,28). Specifically, wild type RmlA was demonstrated to turn over a wide variety of sugar 1-phosphates, including deoxy, amino, and azido sugars, and even displayed weak activity with all eight ribo-and deoxyribonucleotides.…”

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confidence: 99%

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

Moretti

Chang

Peltier‐Pain

et al. 2011

Journal of Biological Chemistry

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Directed evolution is a valuable technique to improve enzyme activity in the absence of a priori structural knowledge, which can be typically enhanced via structure-guided strategies. In this study, a combination of both whole-gene error-prone polymerase chain reaction and site-saturation mutagenesis enabled the rapid identification of mutations that improved RmlA activity toward non-native substrates. These mutations have been shown to improve activities over 10-fold for several targeted substrates, including non-native pyrimidine-and purine-based NTPs as well as non-native D-and L-sugars (both ␣-and ␤-isomers). This study highlights the first broadly applicable high throughput sugar-1-phosphate nucleotidyltransferase screen and the first proof of concept for the directed evolution of this enzyme class toward the identification of uniquely permissive RmlA variants.

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Section: Table 2 Specific Activity Of Selected Sugar Mutantsmentioning

confidence: 99%

mentioning

confidence: 99%

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

Moretti

Chang

Peltier‐Pain

et al. 2011

Journal of Biological Chemistry

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“…In addition, homologous enzymes have been noted to catalyze diverse chemical transformations (15). Recently, the use of protein engineering for altering the reaction specificities of biosynthetic enzymes has emerged as another developing field for modification of natural products (16,17).…”

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confidence: 99%

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

Grocholski

Dinis

Niiranen

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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Bacterial secondary metabolic pathways are responsible for the biosynthesis of thousands of bioactive natural products. Many enzymes residing in these pathways have evolved to catalyze unusual chemical transformations, which is facilitated by an evolutionary pressure promoting chemical diversity. Such divergent enzyme evolution has been observed in S-adenosyl-l-methionine (SAM)-dependent methyltransferases involved in the biosynthesis of anthracycline anticancer antibiotics; whereas DnrK from the daunorubicin pathway is a canonical 4-O-methyltransferase, the closely related RdmB (52% sequence identity) from the rhodomycin pathways is an atypical 10-hydroxylase that requires SAM, a thiol reducing agent, and molecular oxygen for activity. Here, we have used extensive chimeragenesis to gain insight into the functional differentiation of RdmB and show that insertion of a single serine residue to DnrK is sufficient for introduction of the monooxygenation activity. The crystal structure of DnrK-Ser in complex with aclacinomycin T and S-adenosyl-l-homocysteine refined to 1.9-Å resolution revealed that the inserted serine S297 resides in an α-helical segment adjacent to the substrate, but in a manner where the side chain points away from the active site. Further experimental work indicated that the shift in activity is mediated by rotation of a preceding phenylalanine F296 toward the active site, which blocks a channel to the surface of the protein that is present in native DnrK. The channel is also closed in RdmB and may be important for monooxygenation in a solvent-free environment. Finally, we postulate that the hydroxylation ability of RdmB originates from a previously undetected 10-decarboxylation activity of DnrK.

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“…Subtle alteration of the sugars in natural products could change their molecular and cellular specificity, leading to changes in pharmacological properties (16). Glycones are biologically synthesized using nucleotide sugars as a sugar donor (25). Because the number of commercially available nucleotide sugars is limited, the creation of diverse glycosylated secondary metabolites is challenging.…”

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confidence: 99%

Production of a Novel Quercetin Glycoside through Metabolic Engineering of Escherichia coli

Yoon

Kim

Lee

et al. 2012

Appl Environ Microbiol

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ABSTRACTMost flavonoids exist as sugar conjugates. Naturally occurring flavonoid sugar conjugates include glucose, galactose, glucuronide, rhamnose, xylose, and arabinose. These flavonoid glycosides have diverse physiological activities, depending on the type of sugar attached. To synthesize an unnatural flavonoid glycoside,Actinobacillus actinomycetemcomitansgenetll(encoding dTDP-6-deoxy-l-lyxo-4-hexulose reductase, which converts the endogenous nucleotide sugar dTDP-4-dehydro-6-deoxy-l-mannose to dTDP-6-deoxytalose) was introduced intoEscherichia coli. In addition, nucleotide-sugar dependent glycosyltransferases (UGTs) were screened to find a UGT that could use dTDP-6-deoxytalose. Supplementation of this engineered strain ofE. coliwith quercetin resulted in the production of quercetin-3-O-(6-deoxytalose). To increase the production of quercetin 3-O-(6-deoxytalose) by increasing the supplement of dTDP-6-deoxytalose inE. coli, we engineered nucleotide biosynthetic genes ofE. coli, such asgalU(UTP-glucose 1-phosphate uridyltransferase),rffA(dTDP-4-oxo-6-deoxy-d-glucose transaminase), and/orrfbD(dTDP-4-dehydrorahmnose reductase). The engineeredE. colistrain produced approximately 98 mg of quercetin 3-O-(6-deoxytalose)/liter, which is 7-fold more than that produced by the wild-type strain, and the by-products, quercetin 3-O-glucose and quercetin 3-O-rhamnose, were also significantly reduced.

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The impact of enzyme engineering upon natural product glycodiversification

Cited by 90 publications

References 72 publications

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

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

Divergent evolution of an atypical S -adenosyl- l -methionine–dependent monooxygenase involved in anthracycline biosynthesis

Production of a Novel Quercetin Glycoside through Metabolic Engineering of Escherichia coli

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