title: An extra peptide within the catalytic module of a β-agarase affects the agarose degradation pattern.
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 (Asn(246)-Gly(302)) within its GH module. Heterologously expressed AgaG4 (recombinant 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 Tyr(276) 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.
authors: State Key Laboratory of Microbial Technology, School of Life Science, Shandong University, Jinan 250100, China.
authors: Wen-Jun
authors: WJ
authors: Han
authors: Jing-Yan
authors: JY
authors: Gu
authors: Hui-Hui
authors: HH
authors: Liu
authors: Fu-Chuan
authors: FC
authors: Li
authors: Zhi-Hong
authors: ZH
authors: Wu
authors: Yue-Zhong
authors: YZ
authors: Li
mesh_terms: D000595:Amino Acid Sequence; D001419:Bacteria / Q000235:genetics; D002384:Catalysis; D020134:Catalytic Domain; D003001:Cloning, Molecular; D019295:Computational Biology / Q000379:methods; D017931:DNA Primers / Q000235:genetics; D005822:Genetic Vectors; D006026:Glycoside Hydrolases / Q000737:chemistry*; D008957:Models, Genetic; D008969:Molecular Sequence Data; D009154:Mutation; D009844:Oligosaccharides / Q000737:chemistry; D010455:Peptides / Q000737:chemistry*; D010802:Phylogeny; D011485:Protein Binding; D012685:Sepharose / Q000737:chemistry*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D017931:DNA Primers; D009844:Oligosaccharides; D010455:Peptides; D012685:Sepharose; D006026:Glycoside Hydrolases; C019624:agarase
references: Eur J Biochem. 1993 Jun 1;214(2):599-607
references: 8513809
references: Mar Drugs. 2010 Jan 26;8(1):200-18
references: 20161978
references: Adv Carbohydr Chem Biochem. 1969;24:267-332
references: 4913938
references: Int J Syst Evol Microbiol. 2007 Jun;57(Pt 6):1327-1330
references: 17551052
references: Appl Microbiol Biotechnol. 2006 Jun;71(1):23-33
references: 16550377
references: Appl Biochem Biotechnol. 2008 Oct;151(1):51-9
references: 18785021
references: Curr Microbiol. 2005 Apr;50(4):212-6
references: 15902469
references: J Biol Chem. 2003 Nov 21;278(47):47171-80
references: 12970344
references: Appl Environ Microbiol. 2007 Jul;73(14):4691-4
references: 17513582
references: Eur J Biochem. 1983 Dec 1;137(1-2):149-54
references: 6653550
references: J Biol Chem. 2012 Aug 31;287(36):30571-84
references: 22778272
references: Int J Syst Evol Microbiol. 2012 Apr;62(Pt 4):937-941
references: 21669915
references: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W576-81
references: 20542909
references: Glycobiology. 2009 Jun;19(6):615-23
references: 19240276
references: Eur J Biochem. 1983 Oct 3;135(3):553-8
references: 6617649
references: Electrophoresis. 1997 Dec;18(15):2714-23
references: 9504803
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Dec 1;66(Pt 12):1635-9
references: 21139213
references: Appl Environ Microbiol. 2006 Sep;72(9):6399-401
references: 16957270
references: Carbohydr Res. 1994 Feb 3;253:69-77
references: 8156559
references: Int J Syst Evol Microbiol. 2006 Sep;56(Pt 9):2095-2100
references: 16957105
references: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2010 Apr 1;66(Pt 4):413-7
references: 20383011
references: Appl Microbiol Biotechnol. 2012 May;94(4):917-30
references: 22526785
references: Curr Opin Struct Biol. 1997 Oct;7(5):637-44
references: 9345621
references: J Biosci Bioeng. 2002;93(5):456-63
references: 16233232
references: Appl Microbiol Biotechnol. 2012 Feb;93(4):1503-12
references: 21792591
references: J Microbiol Biotechnol. 2011 Nov;21(11):1116-22
references: 22127121
references: Mol Biol Evol. 2011 Oct;28(10):2731-9
references: 21546353
references: Appl Environ Microbiol. 2001 Dec;67(12):5771-9
references: 11722934
references: Anal Biochem. 1995 Sep 20;230(2):229-38
references: 7503412
references: J Biosci Bioeng. 2011 Sep;112(3):225-32
references: 21715227
references: J Biol Chem. 2007 Feb 9;282(6):3747-54
references: 17166842
references: Nature. 2010 Apr 8;464(7290):908-12
references: 20376150
references: Biochem J. 2005 Feb 1;385(Pt 3):703-13
references: 15456406
references: Biotechnol Lett. 2010 Jul;32(7):943-9
references: 20213521
references: Nucleic Acids Res. 1979 Nov 24;7(6):1513-23
references: 388356