Creating Space for Large Acceptors: Rational Biocatalyst Design for Resveratrol Glycosylation in an Aqueous System

Capra

et al. 2019

IJMS

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In family GH13 of the carbohydrate-active enzyme database, subfamily 18 contains glycoside phosphorylases that act on α-sugars and glucosides. Because their phosphorolysis reactions are effectively reversible, these enzymes are of interest for the biocatalytic synthesis of various glycosidic compounds. Sucrose 6F-phosphate phosphorylases (SPPs) constitute one of the known substrate specificities. Here, we report the characterization of an SPP from Ilumatobacter coccineus with a far stricter specificity than the previously described promiscuous SPP from Thermoanaerobacterium thermosaccharolyticum. Crystal structures of both SPPs were determined to provide insight into their similarities and differences. The residues responsible for binding the fructose 6-phosphate group in subsite +1 were found to differ considerably between the two enzymes. Furthermore, several variants that introduce a higher degree of substrate promiscuity in the strict SPP from I. coccineus were designed. These results contribute to an expanded structural knowledge of enzymes in subfamily GH13_18 and facilitate their rational engineering.

Section: Discussionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Structural Comparison of a Promiscuous and a Highly Specific Sucrose 6F-Phosphate Phosphorylase

Capra

et al. 2019

IJMS

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“…Finding such novel enzymes is not only interesting from a fundamental point of view by unveiling new metabolic pathways, but it can also offer new possibilities for practical applications. This was demonstrated in the case of SPP, for which a mutant could glucosylate a far broader range of bulky acceptors than any other SP known to date (8).…”

mentioning

confidence: 85%

Glucosylglycerate Phosphorylase, an Enzyme with Novel Specificity Involved in Compatible Solute Metabolism

Pinel

2017

Appl Environ Microbiol

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Family GH13_18 of the carbohydrate-active enzyme database consists of retaining glycoside phosphorylases that have attracted interest with their potential for synthesizing valuable α-sugars and glucosides. Sucrose phosphorylase was believed to be the only enzyme with specificity in this subfamily for many years, but recent work revealed an enzyme with a different function and hinted at an even broader diversity that is left to discover. In this study, a putative sucrose phosphorylase from that resides in a previously unexplored branch of the family's phylogenetic tree was expressed and characterized. Unexpectedly, no activity on sucrose was observed. Guided by a thorough inspection of the genomic landscape surrounding other genes in the branch, the enzyme was found to be a glucosylglycerate phosphorylase, with a specificity never before reported. Homology modeling, docking, and mutagenesis pinpointed particular acceptor site residues (Asn275 and Glu383) involved in the binding of glycerate. Various organisms known to synthesize and accumulate glucosylglycerate as a compatible solute possess a putative glucosylglycerate phosphorylase gene, indicating that the phosphorylase may be a regulator of its intracellular levels. Moreover, homologs of this novel enzyme appear to be distributed among diverse bacterial phyla, a finding which suggests that many more organisms may be capable of assimilating or synthesizing glucosylglycerate than previously assumed. Glycoside phosphorylases are an intriguing group of carbohydrate-active enzymes that have been used for the synthesis of various economically appealing glycosides and sugars, and they are frequently subjected to enzyme engineering to further expand their application potential. The novel specificity discovered in this work broadens the diversity of these phosphorylases and opens up new possibilities for the efficient production of glucosylglycerate, which is a remarkably potent and versatile stabilizer for protein formulations. Finally, it is a new piece of the puzzle of glucosylglycerate metabolism, being the only known enzyme capable of catalyzing the breakdown of glucosylglycerate in numerous bacterial phyla.

“…Although all above examples were centred around BaSP, other enzymes from GH13_18 can be attractive templates for engineering too. Before the Q345F mutation of BaSP was reported to stimulate the glycosylation of resveratrol, that goal had already been accomplished by redesigning the active site of TtSPP [78]. Despite not being a true SP, TtSPP still shows good affinity for sucrose with the added advantage of already being more active on smaller 'building blocks' of resveratrol, i.e., orcinol and resorcinol.…”

Section: Engineering Of Specificity or Selectivitymentioning

confidence: 99%

Sucrose Phosphorylase and Related Enzymes in Glycoside Hydrolase Family 13: Discovery, Application and Engineering

2020

IJMS

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Sucrose phosphorylases are carbohydrate-active enzymes with outstanding potential for the biocatalytic conversion of common table sugar into products with attractive properties. They belong to the glycoside hydrolase family GH13, where they are found in subfamily 18. In bacteria, these enzymes catalyse the phosphorolysis of sucrose to yield α-glucose 1-phosphate and fructose. However, sucrose phosphorylases can also be applied as versatile transglucosylases for the synthesis of valuable glycosides and sugars because their broad promiscuity allows them to transfer the glucosyl group of sucrose to a diverse collection of compounds other than phosphate. Numerous process and enzyme engineering studies have expanded the range of possible applications of sucrose phosphorylases ever further. Moreover, it has recently been discovered that family GH13 also contains a few novel phosphorylases that are specialised in the phosphorolysis of sucrose 6F-phosphate, glucosylglycerol or glucosylglycerate. In this review, we provide an overview of the progress that has been made in our understanding and exploitation of sucrose phosphorylases and related enzymes over the past ten years.