Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Poulin, Myles B.; Nothaft, Harald; Hug, Isabelle; Feldman, Mario F.; Szymanski, Christine M.; Lowary, Todd L.

doi:10.1074/jbc.m109.072157

Cited by 30 publications

(40 citation statements)

References 60 publications

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“…Previously, it has been shown that coupling glycosyltransferases with the mutase has been effective in overcoming the low yield associated with Gal p to Gal f turnover. 40-43 In this report, a large excess of UDP-Gal p was used, so it is not clear how much this coupling benefits the low turnover of the WcfM reaction. We have now established the complete in vitro reconstitution of the repeat unit of CPSA using key fluorescent isoprenoid probes.…”

Section: Discussionmentioning

confidence: 95%

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

2016

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Capsular polysaccharide A (CPSA) is a four-sugar repeating unit polymer found on the surface of the gut symbiont Bacteroides fragilis that has therapeutic potential in animal models of autoimmune disorders. This therapeutic potential has been credited to its zwitterionic character derived from a positively charged N-acetyl-4-aminogalactosamine (AADGal) and a negatively charged 4,6-O-pyruvylated galactose (PyrGal). In this report, using a fluorescent polyisoprenoid chemical probe, the complete enzymatic assembly of the CPSA tetrasaccharide repeat unit is achieved. The proposed pyruvyltransferase, WcfO, galactopyranose mutase, WcfM, and glycosyltransferases, WcfP and WcfN, encoded by the CPSA biosynthesis gene cluster were heterologously expressed and functionally characterized. Pyruvate modification, catalyzed by WcfO, was found to occur on galactose of the polyisoprenoid-linked disaccharide (AADGal-Gal), and did not occur on galactose linked to uridine diphosphate (UDP) or a set of nitrophenyl-galactose analogues. This pyruvate modification was also found to be required for the incorporation of the next sugar in the pathway N-acetylgalactosamine (GalNAc) by the glycosyltransferase WcfP. The pyruvate acetal modification of a galactose has not been previously explored in the context of a polysaccharide biosynthesis pathway, and this work demonstrates the importance of this modification to repeat unit assembly. Upon production of the polyisoprenoid-linked AADGal-PyrGal-GalNAc, the proteins WcfM and WcfN were found to work in concert to form the final tetrasaccharide, where WcfM formed UDP-galactofuranose (Galf) and WcfN transfers Galf to the AADGal-PyrGal-GalNAc. This work demonstrates the first enzymatic assembly of the tetrasaccharide repeat unit of CPSA in a sequential single pot reaction.

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Section: Discussionmentioning

confidence: 95%

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

2016

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“…It catalyzes a key step in the degradation of both flavonols (via flavanonols) and flavones (via flavanones). Bifunctionality is widespread among bacterial enzymes due to their compact genomes (34)(35)(36)(37)(38). However, CHI is not a classical bifunctional enzyme, as it does not utilize two different domains for catalysis.…”

Section: Discussionmentioning

confidence: 99%

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

et al. 2016

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The enzyme catalyzing the ring-contracting conversion of the flavanonol taxifolin to the auronol alphitonin in the course of flavonoid degradation by the human intestinal anaerobe Eubacterium ramulus was purified and characterized. It stereospecifically catalyzed the isomerization of (؉)-taxifolin but not that of (؊)-taxifolin. The K m for (؉)-taxifolin was 6.4 ؎ 0.8 M, and the V max was 108 ؎ 4 mol min ؊1 (mg protein) ؊1 . The enzyme also isomerized (؉)-dihydrokaempferol, another flavanonol, to maesopsin. Inspection of the encoding gene revealed its complete identity to that of the gene encoding chalcone isomerase (CHI) from E. ramulus. Based on the reported X-ray crystal structure of CHI (M. Gall et al., Angew Chem Int Ed 53:1439 -1442, 2014, http://dx.doi.org/10.1002/anie.201306952), docking experiments suggest the substrate binding mode of flavanonols and their stereospecific conversion. Mutation of the active-site histidine (His33) to alanine led to a complete loss of flavanonol isomerization by CHI, which indicates that His33 is also essential for this activity. His33 is proposed to mediate the stereospecific abstraction of a proton from the hydroxymethylene carbon of the flavanonol C-ring followed by ring opening and recyclization. A flavanonol-isomerizing enzyme was also identified in the flavonoid-converting bacterium Flavonifractor plautii based on its 50% sequence identity to the CHI from E. ramulus. IMPORTANCE Chalcone isomerase was known to be involved in flavone/flavanone conversion by the human intestinal bacterium E. ramulus.Here we demonstrate that this enzyme moreover catalyzes a key step in the breakdown of flavonols/flavanonols. Thus, a single isomerase plays a dual role in the bacterial conversion of dietary bioactive flavonoids. The identification of a corresponding enzyme in the human intestinal bacterium F. plautii suggests a more widespread occurrence of this isomerase in flavonoid-degrading bacteria. F lavonoids represent a major group of plant-derived polyphenolic compounds and have been implicated in beneficial effects on human health (1-4). The flavonoids are classified into several subgroups, which include, among others, flavonols, flavanonols, flavones, flavanones, and chalcones. Quercetin (Fig. 1) is a highly abundant dietary flavonoid that shows a broad range of biological activities. Thus, this flavonol has been intensively studied with respect to its role in disease prevention and use in therapy (5-7). The effects of quercetin and other flavonoids depend on how they are metabolized in the human body, including their conversion by intestinal bacteria (8). Beside deglycosylation and deconjugation, intestinal bacteria are also able to further transform the resulting flavonoid aglycones. However, knowledge about the corresponding bacterial species and, in particular, the enzymes involved is still limited.Eubacterium ramulus and Flavonifractor plautii (formerly Clostridium orbiscindens) are human intestinal bacteria that have been demonstrated to cleave the central heterocyc...

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“…Indeed, examination of the HPLC data revealed that both mutants were capable of interconverting UDP-Glc p /UDP-Gal p . Unfortunately, interconversion for the UDP-Glc p NAc/UDP-Gal p NAc pair by AnGALE was not separable by HPLC and as a result, the reaction was coupled with the enzyme UDP-N-acetylgalactopyranose mutase (UNGM) [69] to form a measurable product (UDP-Gal f NAc). UNGM functions in a similar manner to UGM catalyzing the production of UDP-Gal f NAc from precursor UDP-Gal p NAc.…”

Section: Resultsmentioning

confidence: 99%

Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase

Dalrymple

Sheoran

et al. 2013

PLoS ONE

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The frequency of invasive fungal infections has rapidly increased in recent years. Current clinical treatments are experiencing decreased potency due to severe host toxicity and the emergence of fungal drug resistance. As such, new targets and their corresponding synthetic pathways need to be explored for drug development purposes. In this context, galactofuranose residues, which are employed in fungal cell wall construction, but are notably absent in animals, represent an appealing target. Herein we present the structural and biochemical characterization of UDP-galactose-4-epimerase from Aspergillus nidulans which produces the precursor UDP-galactopyranose required for galactofuranose synthesis. Examination of the structural model revealed both NAD+ and UDP-glucopyranose were bound within the active site cleft in a near identical fashion to that found in the Human epimerase. Mutational studies on the conserved catalytic motif support a similar mechanism to that established for the Human counterpart is likely operational within the A. nidulans epimerase. While the K m and k cat for the enzyme were determined to be 0.11 mM and 12.8 s-1, respectively, a single point mutation, namely L320C, activated the enzyme towards larger N-acetylated substrates. Docking studies designed to probe active site affinity corroborate the experimentally determined activity profiles and support the kinetic inhibition results.

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Characterization of a Bifunctional Pyranose-Furanose Mutase from Campylobacter jejuni 11168

Cited by 30 publications

References 60 publications

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

Complete Tetrasaccharide Repeat Unit Biosynthesis of the Immunomodulatory Bacteroides fragilis Capsular Polysaccharide A

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

Elucidation of Substrate Specificity in Aspergillus nidulans UDP-Galactose-4-Epimerase

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