Thermococcus litoralis 4-␣-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic ␣-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (␤/␣) 7 barrel fold, and a C-terminal domain (domain II), which has a twisted ␤-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu 123 and Asp 214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi ␣-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.

The gene encoding phosphoglucose isomerase was cloned from Thermococcus litoralis, and functionally expressed in Escherichia coli. The puri¢ed enzyme, a homodimer of 21.5 kDa subunits, was biochemically characterized. The inhibition constants for four competitive inhibitors were determined. The enzyme contained 1.25 mol Fe and 0.24 mol Zn per dimer. The activity was enhanced by the addition of Fe , but inhibited by Zn 2+ and EDTA. Enzymes with mutations in conserved histidine and glutamate residues in their cupin motifs contained no metals, and showed large decreases in k cat . The circular dichroism spectra of the mutant enzymes and the wild type enzyme were essentially the same but with slight di¡erences.

Thermococcus litoralis 4-alpha-glucanotransferase (TLGT) belongs to family 57 of glycoside hydrolases and catalyzes the disproportionation and cycloamylose synthesis reactions. Family 57 glycoside hydrolases have not been well investigated, and even the catalytic mechanism involving the active site residues has not been studied. Using 3-ketobutylidene-beta-2-chloro-4-nitrophenyl maltopentaoside (3KBG5CNP) as a donor and glucose as an acceptor, we showed that the disproportionation reaction of TLGT involves a ping-pong bi-bi mechanism. On the basis of this reaction mechanism, the glycosyl-enzyme intermediate, in which a donor substrate was covalently bound to the catalytic nucleophile, was trapped by treating the enzyme with 3KBG5CNP in the absence of an acceptor and was detected by matrix-assisted laser desorption ionization time-of-flight mass spectrometry after peptic digestion. Postsource decay analysis suggested that either Glu-123 or Glu-129 was the catalytic nucleophile of TLGT. Glu-123 was completely conserved between family 57 enzymes, and the catalytic activity of the E123Q mutant enzyme was greatly decreased. On the other hand, Glu-129 was a variable residue, and the catalytic activity of the E129Q mutant enzyme was not decreased. These results indicate that Glu-123 is the catalytic nucleophile of TLGT. Sequence alignment of TLGT and family 38 enzymes (class II alpha-mannosidases) revealed that Glu-123 of TLGT corresponds to the nucleophilic aspartic acid residue of family 38 glycoside hydrolases, suggesting that family 57 and 38 glycoside hydrolases may have had a common ancestor.

This study assessed the effects of Coprinus comatus cap (CCC) on adipogenesis in 3T3-L1 adipocytes and the effects of CCC on the development of diet-induced obesity in rats. Here, we showed that the CCC has an inhibitory effect on the adipocyte differentiation of 3T3-L1 cells, resulting in a significant decrease in lipid accumulation through the downregulation of several adipocyte specific-transcription factors, including CCAAT/enhancer binding protein β, C/EBPδ, and peroxisome proliferator-activated receptor gamma (PPARγ). Moreover, treatment with CCC during adipocyte differentiation induced a significant down-regulation of PPARγ and adipogenic target genes, including adipocyte protein 2, lipoprotein lipase, and adiponectin. Interestingly, the CCC treatment of the 3T3-L1 adipocytes suppressed the insulin-stimulated Akt and GSK3β phosphorylation, and these effects were stronger in the presence of an inhibitor of Akt phosphorylation, LY294002, suggesting that CCC inhibited adipocyte differentiation through the down-regulation of Akt signaling. In the animal study, CCC administration significantly reduced the body weight and adipose tissue weight of rats fed a high fat diet (HFD) and attenuated lipid accumulation in the adipose tissues of the HFD-induced obese rats. The size of the adipocyte in the epididymal fat of the CCC fed rats was significantly smaller than in the HFD rats. CCC treatment significantly reduced the total cholesterol and triglyceride levels in the serum of HFD rats. These results strongly indicated that the CCC-mediated decrease in body weight was due to a reduction in adipose tissue mass. The expression level of PPARγ and phospho-Akt was significantly lower in the CCC-treated HFD rats than that in the HFD obesity rats. These results suggested that CCC inhibited adipocyte differentiation by the down-regulation of major transcription factor involved in the adipogenesis pathway including PPARγ through the regulation of the Akt pathway in 3T3-L1 cells and HFD adipose tissue.