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Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group

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Please use this identifier to cite or link to this item:http://hdl.handle.net/2115/67802

Title: Characterization of Ruminococcus albus cellodextrin phosphorylase and identification of a key phenylalanine residue for acceptor specificity and affinity to the phosphate group
Authors: Sawano, Tatsuya Browse this author
Saburi, Wataru Browse this author →KAKEN DB
Hamura, Ken Browse this author
Matsui, Hirokazu Browse this author →KAKEN DB
Mori, Haruhide Browse this author →KAKEN DB
Keywords: acceptor binding site
cellodextrin phosphorylase
glycoside hydrolase family 94
phosphate binding site
substrate specificity
Issue Date: Sep-2013
Publisher: Wiley-Blackwell
Journal Title: FEBS Journal
Volume: 280
Issue: 18
Start Page: 4463
End Page: 4473
Publisher DOI: 10.1111/febs.12408
PMID: 23802549
Abstract: Ruminococcus albus has the ability to intracellularly degrade cello-oligosaccharides primarily via phosphorolysis. In this study, the enzymatic characteristics of R. albus cellodextrin phosphorylase (RaCDP), which is a member of glycoside hydrolase family 94, was investigated. RaCDP catalyzes the phosphorolysis of cellotriose through an ordered ‘bi bi’ mechanism in which cellotriose binds to RaCDP before inorganic phosphate, and then cellobiose and glucose 1-phosphate (Glc1P) are released in that order. Among the cello-oligosaccharides tested, RaCDP had the highest phosphorolytic and synthetic activities towards cellohexaose and cellopentaose, respectively. RaCDP successively transferred glucosyl residues from Glc1P to the growing cello-oligosaccharide chain, and insoluble cello-oligosaccharides comprising a mean of eight residues were produced. Sophorose, laminaribiose, β-1,4-xylobiose, β-1,4-mannobiose and cellobiitol served as acceptors for RaCDP. RaCDP had very low affinity for phosphate groups in both the phosphorolysis and synthesis directions. A sequence comparison revealed that RaCDP has Gln at position 646 where His is normally conserved in the phosphate binding sites of related enzymes. A Q646H mutant showed approximately twofold lower apparent Km values for inorganic phosphate and Glc1P than the wild-type. RaCDP has Phe at position 633 corresponding to Tyr and Val in the +1 subsites of cellobiose phosphorylase and N,N′-diacetylchitobiose phosphorylase, respectively. A F633Y mutant showed higher preference for cellobiose over β-1,4-mannobiose as an acceptor substrate in the synthetic reaction than the wild-type. Furthermore, the F633Y mutant showed 75- and 1100-fold lower apparent Km values for inorganic phosphate and Glc1P, respectively, in phosphorolysis and synthesis of cellotriose.
Rights: This is the peer reviewed version of the following article: FEBS Journal; 280(18); pp4463-4473; 2013 Sep, which has been published in final form at https://doi.org/10.1111/febs.12408. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.
Type: article (author version)
URI: http://hdl.handle.net/2115/67802
Appears in Collections:農学院・農学研究院 (Graduate School of Agriculture / Faculty of Agriculture) > 雑誌発表論文等 (Peer-reviewed Journal Articles, etc)

Submitter: 佐分利 亘

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