Building mutational bridges between carbohydrate-active enzymes

Franceus, Jorick; Lormans, Jolien; Desmet, Tom

doi:10.1016/j.copbio.2022.102804

Cited by 7 publications

(11 citation statements)

References 34 publications

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“…Positioning tunes the active-site electrostatics and the internuclear distances for bond cleavage/formation. Here, our study emphasizes the subtle level of structural and electronic discrimination that a glycoside hydrolase engineered for reactivity with phosphate − would have to exhibit in order to rival the proficiency of the natural phosphorylase. Finally, the difference in T m of kinetic parameters of the sucrose phosphorylase provides the basis for a discussion of the mechanisms leading to optimum temperature of the enzymatic rates under conditions in which thermal denaturation is still insignificant …”

Section: Discussionmentioning

confidence: 99%

“…Various di- and oligosaccharides, − and even polymeric glycans, ,− have been prepared from glycosyl phosphate substrates using phosphorylase. This has also inspired research into the discovery , and engineering − of GHs that can use phosphate as the nucleophile.…”

Section: Introductionmentioning

confidence: 99%

“…Entropy contributes mostly to substrate binding and appears to involve effects of desolvation at the binding site for the nucleophile/leaving group. Insights into the energetics of the phosphorylase reaction(s) can have relevance for GH catalysis in general and may be of interest in ongoing efforts to replace water by phosphate as nucleophile of enzymatic glycoside conversions. − Among the GHs employing covalent catalysis, the Agrobacterium sp. β-glycosidase , and the E.…”

Section: Introductionmentioning

confidence: 99%

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Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

Vyas

Nidetzky

2023

Biochemistry

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From its structure and mechanism, sucrose phosphorylase is a specialized glycoside hydrolase that uses phosphate ions instead of water as the nucleophile of the reaction. Unlike the hydrolysis reaction, the phosphate reaction is readily reversible and, here, this has enabled the study of temperature effects on kinetic parameters to map the energetic profile of the complete catalytic process via a covalent glycosyl enzyme intermediate. Enzyme glycosylation from sucrose and α-glucose 1-phosphate (Glc1P) is rate-limiting in the forward (k cat = 84 s–1) and reverse direction (k cat = 22 s–1) of reaction at 30 °C. Enzyme–substrate association is driven by entropy (TΔS b ≥ +23 kJ/mol), likely arising from enzyme desolvation at the binding site for the leaving group. Approach from the ES complex to the transition state involves uptake of heat (ΔH ⧧ = 72 ± 5.2 kJ/mol) with little further change in entropy. The free energy barrier for the enzyme-catalyzed glycoside bond cleavage in the substrate is much lower than that for the non-enzymatic reaction (k non), ΔΔG ⧧ = ΔG non ⧧ – ΔG enzyme ⧧ = +72 kJ/mol; sucrose. This ΔΔG ⧧, which also describes the virtual binding affinity of the enzyme for the activated substrate in the transition state (∼1014 M–1), is almost entirely enthalpic in origin. The enzymatic rate acceleration (k cat/k non) is ∼1012-fold and similar for reactions of sucrose and Glc1P. The 103-fold lower reactivity (k cat/K m) of glycerol than fructose in enzyme deglycosylation reflects major losses in the activation entropy, suggesting a role of nucleophile/leaving group recognition by the enzyme in inducing the active-site preorganization required for optimum transition state stabilization by enthalpic forces.

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Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

Vyas

Nidetzky

2023

Biochemistry

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“…These results are interesting in demonstrating the catalytic promiscuity of β-RFAS in glycoside bond formation. Somewhat reminiscent of sugar nucleotide-dependent C -glycosyltransferases that form a C -, N -, or O -glycoside product dependent on the structure of the acceptor substrate used, ,− ForT can also catalyze C–C and C–N bond formation in the β-riboside product released. As in C -glycosyltransferases, the type of product formed appears to be dictated by the acceptor substrate and its accommodation in the binding pocket for the enzyme rather than by the catalytic machinery of the active site.…”

Section: -(D-ribofuranosyl)aminobenzene Synthases (β-Rfass)mentioning

confidence: 99%

C-Ribosylating Enzymes in the (Bio)Synthesis of C-Nucleosides and C-Glycosylated Natural Products

Pfeiffer,

Nidetzky

2023

ACS Catal.

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“…The most common and most extensively studied CAZymes are glycoside hydrolases (GHs) and glycosyltransferases (GTs), which catalyze the breakdown and synthesis of glycosidic bonds, respectively. Among the more peculiar products of CAZyme evolution are the glycoside phosphorylases (GPs), which appear to combine the characteristics of GHs and GTs . GPs have evolved to degrade glycosidic bonds using inorganic phosphate instead of water as their nucleophile of choice, resulting in the release of a glycosyl phosphate.…”

Section: Introductionmentioning

confidence: 99%

Evolution of Phosphorylase Activity in an Ancestral Glycosyltransferase

Franceus,

Rivas-Fernández,

Lormans

et al. 2024

ACS Catal.

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The reconstruction of ancestral sequences can offer a glimpse into the fascinating process of molecular evolution by exposing the adaptive pathways that shape the proteins found in nature today. Here, we track the evolution of the carbohydrate-active enzymes responsible for the synthesis and turnover of mannogen, a critical carbohydrate reserve in Leishmania parasites. Biochemical characterization of resurrected enzymes demonstrated that mannoside phosphorylase activity emerged in an ancestral bacterial mannosyltransferase, and later disappeared in the process of horizontal gene transfer and gene duplication in Leishmania. By shuffling through plausible historical sequence space in an ancestral mannosyltransferase, we found that mannoside phosphorylase activity could be toggled on through various combinations of mutations at positions outside of the active site. Molecular dynamics simulations showed that such mutations can affect loop rigidity and shield the active site from water molecules that disrupt key interactions, allowing α-mannose 1-phosphate to adopt a catalytically productive conformation. These findings highlight the importance of subtle distal mutations in protein evolution and suggest that the vast collection of natural glycosyltransferases may be a promising source of engineering templates for the design of tailored phosphorylases.

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Building mutational bridges between carbohydrate-active enzymes

Cited by 7 publications

References 34 publications

Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

Energetics of the Glycosyl Transfer Reactions of Sucrose Phosphorylase

C-Ribosylating Enzymes in the (Bio)Synthesis of C-Nucleosides and C-Glycosylated Natural Products

Evolution of Phosphorylase Activity in an Ancestral Glycosyltransferase

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