Clostridium thermocellumXyn10B Carbohydrate-Binding Module 22-2:  The Role of Conserved Amino Acids in Ligand Binding†,‡

Biochemistry ◽  
2001 ◽  
Vol 40 (31) ◽  
pp. 9167-9176 ◽  
Author(s):  
Hefang Xie ◽  
Harry J. Gilbert ◽  
Simon J. Charnock ◽  
Gideon J. Davies ◽  
Michael P. Williamson ◽  
...  
Keyword(s):  
Amino Acids ◽  
Ligand Binding ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Conserved Amino Acids

Hydrolysis of softwood by Aspergillus mannanase: Role of a carbohydrate-binding module

2010 ◽  
Vol 148 (4) ◽  
pp. 163-170 ◽  
Author(s):  
Tuan Anh Pham ◽  
Jean Guy Berrin ◽  
Eric Record ◽  
Kim Anh To ◽  
Jean-Claude Sigoillot
Keyword(s):  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Hydrolysis Of

scholarly journals Restoration of high-level transport activity by human reduced folate carrier/ThTr1 thiamine transporter chimaeras: role of the transmembrane domain 6/7 linker region in reduced folate carrier function

2003 ◽  
Vol 369 (1) ◽  
pp. 31-37 ◽  
Author(s):  
Xiang Y. LIU ◽  
Teah L. WITT ◽  
Larry H. MATHERLY
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Plasma Membranes ◽  
Transmembrane Domain ◽  
K562 Cells ◽  
Reduced Folate Carrier ◽  
Wild Type ◽  
Linker Region ◽  
Conserved Amino Acids

The reduced folate carrier (RFC; SLC19A1) is closely related to the thiamine transporter, SLC19A2 (ThTr1). Hydropathy models for these homologous transporters predict up to 12 transmembrane domains (TMDs), with internally oriented N- and C-termini and a large central loop between TMDs 6 and 7. The homologies are localized mostly in the TMDs. However, there is little similarity in their N- and C-terminal domains and the central peptide linkers connecting putative TMDs 1—6 and TMDs 7—12. To explore the functional role of the 61-amino acid central linker in the human RFC (hRFC), we introduced deletions of 49 and 60 amino acids into this region, differing by the presence of a stretch of 11 highly conserved amino acids between the human and rodent RFCs (positions 204—214). An additional hRFC construct was prepared in which only the 11 conserved amino acids were deleted. The resulting hRFCD215—R263Δ, hRFCK204—R263Δ and hRFCK204—R214Δ proteins were transfected into transport-impaired K562 cells. The deletion constructs were all expressed in plasma membranes; however, they were completely inactive for methotrexate and (6S)5-formyl tetrahydrofolate transport. Insertion of non-homologous 73- and 84-amino acid fragments from the structurally analogous ThTr1 linker region into position 204 of hRFCK204—R263Δ restored low levels of transport (16—21% of the wild type). Insertion of the ThTr1 linkers into hRFCD215—R263Δ at position 215 restored 60—80% of wild-type levels of transport. Collectively, our results suggest that the role of the hRFC linker peptide is to provide the proper spatial orientation between the two halves of the hRFC protein for optimal function, and that this is largely independent of amino acid sequence. Our results also demonstrate a critical transport role for the stretch of 11 conserved amino acids starting at position 204 of hRFC.

scholarly journals Novel xylan-binding properties of an engineered family 4 carbohydrate-binding module

2007 ◽  
Vol 406 (2) ◽  
pp. 209-214 ◽  
Author(s):  
Lavinia Cicortas Gunnarsson ◽  
Cedric Montanier ◽  
Richard B. Tunnicliffe ◽  
Mike P. Williamson ◽  
Harry J. Gilbert ◽  
...  
Keyword(s):  
Ligand Binding ◽  
Binding Site ◽  
Hydrophobic Interactions ◽  
Molecular Engineering ◽  
Carbohydrate Binding ◽  
Carbohydrate Binding Module ◽  
Wild Type ◽  
Rhodothermus Marinus ◽  
Binding Properties ◽  
Carbohydrate Binding Modules

Molecular engineering of ligand-binding proteins is commonly used for identification of variants that display novel specificities. Using this approach to introduce novel specificities into CBMs (carbohydrate-binding modules) has not been extensively explored. Here, we report the engineering of a CBM, CBM4-2 from the Rhodothermus marinus xylanase Xyn10A, and the identification of the X-2 variant. As compared with the wild-type protein, this engineered module displays higher specificity for the polysaccharide xylan, and a lower preference for binding xylo-oligomers rather than binding the natural decorated polysaccharide. The mode of binding of X-2 differs from other xylan-specific CBMs in that it only has one aromatic residue in the binding site that can make hydrophobic interactions with the sugar rings of the ligand. The evolution of CBM4-2 has thus generated a xylan-binding module with different binding properties to those displayed by CBMs available in Nature.

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