title: N-Linked glycans are an important component of the processive machinery of cellobiohydrolases.
abstract: Cellobiohydrolases (CBHs), belonging to glycoside hydrolase families 6 and 7 (GH6 and GH7), are the major components of cellulase systems of filamentous fungi involved in biodegradation of cellulose in nature. Previous studies demonstrated that N-linked glycans in the catalytic domains of GH7 CBHs significantly affect the enzyme activity against cellulosic substrates. The influence of N-linked glycans on the activity and processivity of recombinant GH6 CBH II from Penicillium verruculosum (PvCel6A) was studied using site-directed mutagenesis of the respective Asn residues. Depending on the position of N-glycans on the surface of a protein globule, they affected the enzyme activity against cellulose either negatively or positively. The decrease or increase in the degree of processivity of recombinant forms of PvCel6A generally correlated with activity changes against Avicel. The mechanism of the N-glycan influence seems to be universal for GH6 and GH7 CBHs. The observed effects for CBHs from both families are explained in terms of a mechanistic model that also makes clear our previously published data on the highly active CBH IIb from Myceliophthora thermophila (MtCel6B). This study, together with data of other researchers, strongly suggests that the N-linked glycans in the catalytic domains of GH6 and GH7 CBHs are involved in processive catalytic machinery of these enzymes. Data obtained should be taken into account during development of new and more effective biocatalysts by protein engineering techniques.
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia; Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia. Electronic address: avgusakov@enzyme.chem.msu.ru.
authors.forename: Alexander V
authors.initials: AV
authors.lastname: Gusakov
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia; Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Anna S
authors.initials: AS
authors.lastname: Dotsenko
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia; Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Aleksandra M
authors.initials: AM
authors.lastname: Rozhkova
authors.affiliation: Department of Chemistry, M. V. Lomonosov Moscow State University, Vorobyovy Gory 1/11, Moscow 119991, Russia; Federal Research Centre "Fundamentals of Biotechnology", Russian Academy of Sciences, Leninsky Pr. 33, Moscow 119071, Russia.
authors.forename: Arkady P
authors.initials: AP
authors.lastname: Sinitsyn
mesh_terms: D000595:Amino Acid Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D002482:Cellulose / Q000378:metabolism; D043366:Cellulose 1,4-beta-Cellobiosidase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D006031:Glycosylation; D008958:Models, Molecular; D009154:Mutation; D010407:Penicillium / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D011134:Polysaccharides / Q000378:metabolism*; D000072417:Protein Domains; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D017386:Sequence Homology, Amino Acid
chemical_list: D005656:Fungal Proteins; D011134:Polysaccharides; D011994:Recombinant Proteins; D002482:Cellulose; D043366:Cellulose 1,4-beta-Cellobiosidase
keywords: Cellobiohydrolase; Cloning; N-glycosylation; Processivity; Site-directed mutagenesis