Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/<i>N</i>-Acetyltransferase from <i>Shewanella denitrificans</i>

Chantigian, Daniel; Thoden, James B.; Holden, Hazel M.

doi:10.1021/bi401170t

Cited by 14 publications

(26 citation statements)

References 29 publications

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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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Section: Discussionmentioning

confidence: 99%

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

et al. 2016

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“…However, the three-dimensional structure of a bifunctional ketoisomerase/ N -acetyltransferase, referred to as FdtD, was known from our recent research. 11 FdtD, like the A. thermoaerophilus FdtA, is a “galacto” enzyme. 11 Given the overall structure of the bifunctional FdtD, it was possible to clone and express only its 3,4-ketoisomerase domain, which extends from Ser 160 to Asn 304.…”

Section: Resultsmentioning

confidence: 99%

“…11 FdtD, like the A. thermoaerophilus FdtA, is a “galacto” enzyme. 11 Given the overall structure of the bifunctional FdtD, it was possible to clone and express only its 3,4-ketoisomerase domain, which extends from Ser 160 to Asn 304. To ensure that the isolated domain retained catalytic activity, its kinetic parameters were determined and are listed in Table 1.…”

Section: Resultsmentioning

confidence: 99%

“…The catalytic efficiency of the isolated 3,4-ketoisomerase domain is 6.1 × 10 3 s −1 M −1 . When it is part of the bifunctional enzyme, the catalytic efficiency is 9.8 × 10 4 s −1 M −1 11. …”

Section: Resultsmentioning

confidence: 99%

“…11 Specifically, enzymatic activities were measured by monitoring the decrease in absorbance at 340 nm as NADPH is oxidized to NADP + due to the action of KijD10. Previous studies from the laboratory have verified that KijD10 from Actinomadura kijaniata functions as an NADPH-dependent C-3′ ketoreductase.…”

Section: Methodsmentioning

confidence: 99%

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Bacterial Sugar 3,4-Ketoisomerases: Structural Insight into Product Stereochemistry

et al. 2015

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3-Acetamido-3,6-dideoxy-D-galactose (Fuc3NAc) and 3-acetamido-3,6-dideoxy-D-glucose (Qui3NAc) are unusual sugars found on the lipopolysaccharides of Gram-negative bacteria and on the S-layers of Gram-positive bacteria. The 3,4-ketoisomerases, referred to as FdtA and QdtA, catalyze the third steps in the respective biosynthetic pathways for these sugars. Whereas both enzymes utilize the same substrate, the stereochemistries of their products are different. Specifically, the hydroxyl groups at the hexose C-4′ positions assume the “galactose” and “glucose” configurations in the FdtA and QdtA products, respectively. In 2007 we reported the structure of the apoform of FdtA from Aneurinibacillus thermoaerophilus, which was followed in 2014 by the X-ray analysis of QdtA from Thermoanaerobacterium thermosaccharolyticum as a binary complex. Both of these enzymes belong to the cupin superfamily. Here we report a combined structural and enzymological study to explore the manner in which these enzymes control the stereochemistry of their products. Various site-directed mutant proteins of each enzyme were constructed, and their dTDP-sugar products were analyzed by NMR spectroscopy. In addition, the kinetic parameters for these protein variants were measured, and the structure of one, namely, the QdtA Y17R/R97H double mutant form, was determined to 2.3-Å resolution. Finally, in an attempt to obtain a model of FdtA with a bound dTDP-linked sugar, the 3,4-ketoisomerase domain of a bifunctional enzyme from Shewanella denitrificans was cloned, purified, and crystallized in the presence of a dTDP-linked sugar analogue. Taken together, the results from this investigation demonstrate that it is possible to convert a “galacto” enzyme into a “gluco” enzyme and vice versa.

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The molecular architecture of QdtA, a sugar 3,4‐ketoisomerase from Thermoanaerobacterium thermosaccharolyticum

Thoden

Holden²

2014

Protein Science

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Unusual di-and trideoxysugars are often found on the O-antigens of Gram-negative bacteria, on the S-layers of Gram-positive bacteria, and on various natural products. One such sugar is 3-acetamido-3,6-dideoxy-D-glucose. A key step in its biosynthesis, catalyzed by a 3,4-ketoisomerase, is the conversion of thymidine diphosphate (dTDP)24-keto-6-deoxyglucose to dTDP-3-keto-6-deoxyglucose. Here we report an X-ray analysis of a 3,4-ketoisomerase from Thermoanaerobacterium thermosaccharolyticum. For this investigation, the wild-type enzyme, referred to as QdtA, was crystallized in the presence of dTDP and its structure solved to 2.0-Å resolution. The dimeric enzyme adopts a three-dimensional architecture that is characteristic for proteins belonging to the cupin superfamily. In order to trap the dTDP-4-keto-6-deoxyglucose substrate into the active site, a mutant protein, H51N, was subsequently constructed, and the structure of this protein in complex with the dTDP-sugar ligand was solved to 1.9-Å resolution. Taken together, the structures suggest that His 51 serves as a catalytic base, that Tyr 37 likely functions as a catalytic acid, and that His 53 provides a proton shuttle between the C-3 0 hydroxyl and the C-4 0 keto group of the hexose. This study reports the first three-dimensional structure of a 3,4-ketoisomerase in complex with its dTDP-sugar substrate and thus sheds new molecular insight into this fascinating class of enzymes.

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Structural and Biochemical Characterization of a Bifunctional Ketoisomerase/N-Acetyltransferase from Shewanella denitrificans

Cited by 14 publications

References 29 publications

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

Chalcone Isomerase from Eubacterium ramulus Catalyzes the Ring Contraction of Flavanonols

Bacterial Sugar 3,4-Ketoisomerases: Structural Insight into Product Stereochemistry

The molecular architecture of QdtA, a sugar 3,4‐ketoisomerase from Thermoanaerobacterium thermosaccharolyticum

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