Han Wen-Jun, Gu Jing-Yan, Liu Hui-Hui, Li Fu-Chuan, Wu Zhi-Hong, Li Yue-Zhong.

The Journal of Biological Chemistry  2013, 288(13): 9519-9531

ABSTRACT

Agarase hydrolyzes agarose into a series of oligosaccharides with repeating disaccharide units. The glycoside hydrolase (GH) module of agarase is known to be responsible for its catalytic activity. However, variations in the composition of the GH module and its effects on enzymatic functions have been minimally elucidated. The agaG4 gene, cloned from the genome of the agarolytic Flammeovirga strain MY04, encodes a 503-amino acid protein AgaG4. Compared with elucidated agarases, AgaG4 contains an extra peptide (N246-G302) within its GH module. Heterologously expressed AgaG4 (rAgaG4) was determined to be an endo-type β-agarase. The protein degraded agarose into neoagarotetraose and neoagarohexaose at a final molar ratio of 1.5: 1. Neoagarooctaose was the smallest substrate for rAgaG4, whereas neoagarotetraose was the minimal degradation product. Removing the extra fragment from the GH module led to the inability of the mutant (rAgaG4-T57) to degrade neoagarooctaose, and the final degradation products of agarose by the truncated protein were neoagarotetraose, neoagarohexaose, and neoagarooctaose at a final molar ratio of 2.7: 2.8: 1. The optimal temperature for agarose degradation also decreased to 40°C for this mutant. Bioinformatic analysis suggested that tyrosine 276 within the extra fragment was a candidate active site residue for the enzymatic activity. Site-swapping experiments of Tyr276 to 19 various other amino acids demonstrated that the characteristics of this residue were crucial for the AgaG4 degradation of agarose and the cleavage pattern of substrate.