Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Meints, Lisa M.; Poston, Anne W.; Piligian, Brent F.; Olson, Claire D.; Badger, Katherine S.; Woodruff, Peter J.; Swarts, Benjamin M.

doi:10.3791/54485

Cited by 11 publications

(26 citation statements)

References 21 publications

(41 reference statements)

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“…Recombinant expression of TtTreT from the pET302 plasmid in E. coli BL21(DE3) was previously reported to lead to the formation of insoluble inclusion bodies (IBs) (21). Therefore, TtTreT was expressed using the pBAD/His A plasmid in E. coli Top10 based on previous results, where the formation of insoluble inclusion bodies was not reported (40). The protein solubility of TreT was evaluated in the expression host E. coli Top10 in parallel expression experiments using the pBAD/His A vector harboring an empty plasmid or the genes encoding TtTreT, TuTreT, or PyTreT.…”

Section: Resultsmentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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LeLoir glycosyltransferases are important biocatalysts for the production of glycosidic bonds in natural products, chiral building blocks, and pharmaceuticals. Trehalose transferase (TreT) is of particular interest since it catalyzes the stereo-and enantioselective ␣,␣-(1¡1) coupling of a nucleotide sugar donor and monosaccharide acceptor for the synthesis of disaccharide derivatives. Heterologously expressed thermophilic trehalose transferases were found to be intrinsically aggregation prone and are mainly expressed as catalytically active inclusion bodies in Escherichia coli. To disfavor protein aggregation, the thermostable protein mCherry was explored as a fluorescent protein tag. The fusion of mCherry to trehalose transferase from Pyrobaculum yellowstonensis (PyTreT) demonstrated increased protein solubility. Chaotropic agents like guanidine or the divalent cations Mn(II), Ca(II), and Mg(II) enhanced the enzyme activity of the fusion protein. The thermodynamic equilibrium constant, K eq , for the reversible synthesis of trehalose from glucose and a nucleotide sugar was determined in both the synthesis and hydrolysis directions utilizing UDPglucose and ADP-glucose, respectively. UDP-glucose was shown to achieve higher conversions than ADP-glucose, highlighting the importance of the choice of nucleotide sugars for LeLoir glycosyltransferases under thermodynamic control. IMPORTANCE The heterologous expression of proteins in Escherichia coli is of great relevance for their functional and structural characterization and applications. However, the formation of insoluble inclusion bodies is observed in approximately 70% of all cases, and the subsequent effects can range from reduced soluble protein yields to a complete failure of the expression system. Here, we present an efficient methodology for the production and analysis of a thermostable, aggregation-prone trehalose transferase (TreT) from Pyrobaculum yellowstonensis via its fusion with mCherry as a thermostable fluorescent protein tag. This fusion strategy allowed for increased enzyme stability and solubility and could be applied to other (thermostable) proteins, allowing rapid visualization and quantification of the mCherry-fused protein of interest. Finally, we have demonstrated that the enzymatic synthesis of trehalose from glucose and a nucleotide sugar is reversible by approaching the thermodynamic equilibrium in both the synthesis and hydrolysis directions. Our results show that uridine establishes an equilibrium constant which is more in favor of the product trehalose than when adenosine is employed as the nucleotide under identical conditions. The influence of different nucleotides on the reaction can be generalized for all LeLoir glycosyltransferases under thermodynamic control as the position of the equilibrium depends solely on the reaction conditions and is not affected by the nature of the catalyst.

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Section: Resultsmentioning

confidence: 99%

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Mestrom

Marsden

Dieters

et al. 2019

Appl Environ Microbiol

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“…Accordingly, SIRT1 overexpression was insufficient to upregulate ALOXE3 in vivo. It is therefore intriguing that we were able to achieve enhanced in vitro ALOXE3 upregulation comparable to viral ALOXE3 overexpression by treatment with the trehalase-resistant trehalose analogue, lactotrehalose (22)(23)(24)50,51). Our characterization of this glucose-galactose trehalose analogue, and the finding that lactotrehalose exhibits enhanced fastingmimetic potency is a critical advance for at least two reasons.…”

Section: Discussionmentioning

confidence: 97%

ALOXE3 is a hepatic fasting-responsive lipoxygenase that enhances insulin sensitivity via hepatic PPARγ

Higgins

Zhang

et al. 2018

Preprint

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The hepatic glucose fasting response is gaining traction as a therapeutic pathway to enhance hepatic and wholehost metabolism. However, the mechanisms underlying these metabolic effects remain unclear. Here, we demonstrate the lipoxygenase, ALOXE3, is a novel effector of the therapeutic fasting response. We show that ALOXE3 is activated during fasting, glucose withdrawal, or trehalose/trehalose analogue treatment. Hepatocytespecific ALOXE3 expression reduced weight gain and hepatic steatosis in diet-induced and genetically obese (db/db) models. ALOXE3 expression moreover enhanced basal thermogenesis and abrogated insulin resistance in db/db diabetic mice. Targeted metabolomics demonstrated accumulation of the PPARg ligand, 12-KETE in hepatocytes overexpressing ALOXE3. Strikingly, PPARg inhibition reversed hepatic ALOXE3-mediated insulin sensitization, suppression of hepatocellular ATP production and oxygen consumption, and gene induction of PPARg coactivator-1a (PGC1a) expression. Moreover, hepatocyte-specific PPARg deletion reversed the therapeutic effect of hepatic ALOXE3 expression on diet-induced insulin intolerance. ALOXE3 is therefore a novel effector of the hepatocellular fasting response that leverages both PPARg-mediated and pleiotropic effects to augment hepatic and whole-host metabolism, and is thus a promising target to ameliorate metabolic disease.

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“…Accordingly, SIRT1 overexpression was insufficient to upregulate Aloxe3 in vivo. It is therefore intriguing that we were able to achieve enhanced in vitro Aloxe3 upregulation comparable with viral Aloxe3 overexpression by treatment with the trehalase-resistant trehalose analogue lactotrehalose ( 21 – 23 , 54 , 55 ). Our characterization of this glucose-galactose trehalose analogue, and the finding that lactotrehalose exhibits enhanced fasting-mimetic potency, is a critical advance for at least 2 reasons.…”

Section: Discussionmentioning

confidence: 99%

Hepatocyte ALOXE3 is induced during adaptive fasting and enhances insulin sensitivity by activating hepatic PPARγ

Higgins¹,

Zhang²,

Mayer³

et al. 2018

JCI Insight

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The hepatic glucose fasting response is gaining traction as a therapeutic pathway to enhance hepatic and whole-host metabolism. However, the mechanisms underlying these metabolic effects remain unclear. Here, we demonstrate the epidermal-type lipoxygenase, eLOX3 (encoded by its gene, Aloxe3), is a potentially novel effector of the therapeutic fasting response. We show that Aloxe3 is activated during fasting, glucose withdrawal, or trehalose/trehalose analogue treatment. Hepatocyte-specific Aloxe3 expression reduced weight gain and hepatic steatosis in diet-induced and genetically obese (db/db) mouse models. Aloxe3 expression, moreover, enhanced basal thermogenesis and abrogated insulin resistance in db/db diabetic mice. Targeted metabolomics demonstrated accumulation of the PPARγ ligand 12-KETE in hepatocytes overexpressing Aloxe3. Strikingly, PPARγ inhibition reversed hepatic Aloxe3–mediated insulin sensitization, suppression of hepatocellular ATP production and oxygen consumption, and gene induction of PPARγ coactivator-1α (PGC1α) expression. Moreover, hepatocyte-specific PPARγ deletion reversed the therapeutic effect of hepatic Aloxe3 expression on diet-induced insulin intolerance. Aloxe3 is, therefore, a potentially novel effector of the hepatocellular fasting response that leverages both PPARγ-mediated and pleiotropic effects to augment hepatic and whole-host metabolism, and it is, thus, a promising target to ameliorate metabolic disease.

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Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

Cited by 11 publications

References 21 publications

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

Artificial Fusion of mCherry Enhances Trehalose Transferase Solubility and Stability

ALOXE3 is a hepatic fasting-responsive lipoxygenase that enhances insulin sensitivity via hepatic PPARγ

Hepatocyte ALOXE3 is induced during adaptive fasting and enhances insulin sensitivity by activating hepatic PPARγ

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