Studies on a hyperthermostable 4-α-glucanotransferase: X-ray structure analysis and enzymatic reaction mechanism

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4-α-Glucanotransferase (Gtase) of a hyperthermophilic archaeon, Thermococcus litoralis, synthesizes linear and cyclic α-I ,4-glucans from oligomaltose and amylose as substrates. Following results were obtained in this research.(1) The Gtase gene has a high content of AGA and AGG codons for arginine, which are extremely rare in Escherichia coli. Expression of the gene in E. coli resulted in low production of the enzyme and the accumulation of inclusion bodies. However, simultaneous expression of Gtase with tRNAィイD2AGAィエD2, tRNAィイD2AGGィエD2 and GroELS affected both the production and solubility of the enzyme, and production of soluble Gtase increased about 5-fold.(2) This enzyme reaction was carried out through the Ping-Pong BiBi mechanism, when analyzed with substrates, 3-ketobutylidene-β-2-chloro-4-nitrophenyl-maltopentaoside (donor) and glucose (acceptor).(3) This Gtase belongs to the family 57 of glycosyl hydrolases. No active site residues are identified in the family 57 enzymes. Comparing the amino acid sequences, 8 carboxylic resides were conserved in the family. Analyses of the mutant enzymes constructed by site-directed mutagenesis suggested that Gln 123, Asp 145, Asp2l4, Glu216, and Asp354 are present in or around the active site.(4) Crystals of the enzyme were obtained by using ammonium sulfate and PEG400 as precipitants. The space group was P6ィイD24ィエD222, and the unit cell dimensions were a=b= 125 Å and c=246 Å.