title: Molecular insights into substrate specificity and thermal stability of a bacterial GH5-CBM27 endo-1,4-β-D-mannanase.
abstract: The breakdown of β-1,4-mannoside linkages in a variety of mannan-containing polysaccharides is of great importance in industrial processes such as kraft pulp delignification, food processing and production of second-generation biofuels, which puts a premium on studies regarding the prospection and engineering of β-mannanases. In this work, a two-domain β-mannanase from Thermotoga petrophila that encompasses a GH5 catalytic domain with a C-terminal CBM27 accessory domain, was functionally and structurally characterized. Kinetic and thermal denaturation experiments showed that the CBM27 domain provided thermo-protection to the catalytic domain, while no contribution on enzymatic activity was observed. The structure of the catalytic domain determined by SIRAS revealed a canonical (α/β)(8)-barrel scaffold surrounded by loops and short helices that form the catalytic interface. Several structurally related ligand molecules interacting with TpMan were solved at high-resolution and resulted in a wide-range representation of the subsites forming the active-site cleft with residues W134, E198, R200, E235, H283 and W284 directly involved in glucose binding.
authors.affiliation: Laboratório Nacional de Biociências, Centro Nacional de Pesquisa em Energia e Materiais, Campinas, SP, Brazil.
authors.forename: Camila Ramos
authors.initials: CR
authors.lastname: dos Santos
authors.forename: Joice Helena
authors.initials: JH
authors.lastname: Paiva
authors.forename: Andreia Navarro
authors.initials: AN
authors.lastname: Meza
authors.forename: Junio
authors.initials: J
authors.lastname: Cota
authors.forename: Thabata Maria
authors.initials: TM
authors.lastname: Alvarez
authors.forename: Roberto
authors.initials: R
authors.lastname: Ruller
authors.forename: Rolf Alexander
authors.initials: RA
authors.lastname: Prade
authors.forename: Fabio Marcio
authors.initials: FM
authors.lastname: Squina
authors.forename: Mario Tyago
authors.initials: MT
authors.lastname: Murakami
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004795:Enzyme Stability; D005947:Glucose / Q000737:chemistry; D016965:Gram-Negative Anaerobic Straight, Curved, and Helical Rods / Q000201:enzymology*; D007700:Kinetics; D008320:Maltose / Q000737:chemistry; D008361:Mannosidases / Q000737:chemistry* / Q000235:genetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011485:Protein Binding; D011489:Protein Denaturation; D017384:Sequence Deletion; D013379:Substrate Specificity; D013499:Surface Properties
chemical_list: D001426:Bacterial Proteins; D008320:Maltose; D008361:Mannosidases; C041867:endo-1,4-beta-D-mannanase; D005947:Glucose