Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,<i>Thermotoga maritima</i>MSB8

Rajashekhara, Eranna; Kitaoka, Motomitsu; Kim, Yeon-Kye; Hayashi, Kiyoshi

doi:10.1271/bbb.66.2578

Cited by 42 publications

(40 citation statements)

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“…CbP and CdP have been purified from C. thermocellum (4)(5)(6)(7)40), and the presence of one or both of these enzymes has also been documented in other cellulolytic bacteria, including Ruminococcus flavefaciens (9), C. gilvus (36), R. albus (27), Prevotella ruminicola (26), C. stercorarium (34) and Thermotoga neapolitana (45), and T. maritime (33). Although C. thermocellum grows at ϳ60°C, specific activities of CbP and CdP have been measured at 37°C in prior studies (6)(7)(8)43).…”

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confidence: 99%

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Zhang¹,

Lynd

2004

Appl Environ Microbiol

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Rates of phosphorolytic cleavage of ␤-glucan substrates were determined for cell extracts from Clostridium thermocellum ATCC 27405 and were compared to rates of hydrolytic cleavage. Reactions with cellopentaose and cellobiose were evaluated for both cellulose (Avicel)-and cellobiose-grown cultures, with more limited data also obtained for cellotetraose. To measure the reaction rate in the chain-shortening direction at elevated temperatures, an assay protocol was developed featuring discrete sampling at 60°C followed by subsequent analysis of reaction products (glucose and glucose-1-phosphate) at 35°C. Calculated rates of phosphorolytic cleavage for cell extract from Avicel-grown cells exceeded rates of hydrolytic cleavage by >20-fold for both cellobiose and cellopentaose over a 10-fold range of ␤-glucan concentrations (0.5 to 5 mM) and for cellotetraose at a single concentration (2 mM). Rates of phosphorolytic cleavage of ␤-glucosidic bonds measured in cell extracts were similar to rates observed in growing cultures. Comparisons of V max values indicated that cellobiose-and cellodextrin-phosphorylating activities are synthesized during growth on both cellobiose and Avicel but are subject to some degree of metabolic control. The apparent K m for phosphorolytic cleavage was lower for cellopentaose (mean value for Avicel-and cellobiose-grown cells, 0.61 mM) than for cellobiose (mean value, 3.3 mM).

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confidence: 99%

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Zhang¹,

Lynd

2004

Appl Environ Microbiol

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“…were reported on the same acceptor [25]. The biggest difference is observed in the K m value, which is 2.4-fold lower for the E649C mutant.…”

Section: Discussionmentioning

confidence: 69%

“…The kinetic parameters of the C. uda CP E649C variant on methyl β-glucoside are better than those of Thermotoga maritima CP, which is the only known CP active on methyl β-glucoside [25]. For this enzyme a K m value of 135 mM and a k cat value of 6.5 s…”

Section: Discussionmentioning

confidence: 90%

Development and application of a screening assay for glycoside phosphorylases

Groeve

Tran

Hoorebeke

et al. 2010

Analytical Biochemistry

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Glycoside phosphorylases (GPs) are interesting enzymes for the glycosylation of chemical molecules. They only require a glycosyl phosphate as sugar donor and an acceptor molecule with a free hydroxyl group. Their narrow substrate specificity, however, limits the application of GPs for general glycoside synthesis. Although an enzyme's substrate specificity can be altered and broadened by protein engineering and directed evolution, this requires a suitable screening assay. Such a screening assay has not yet been described for GPs. Here, we report a screening procedure for GPs based on the measurement of released inorganic phosphate in the direction of glycoside synthesis. It appeared necessary to inhibit endogenous phosphatase activity in crude Escherichia coli cell extracts with molybdate, and inorganic phosphate was measured with a modified phosphomolybdate method. The screening system is general and can be used to screen GP enzyme libraries for novel donor and acceptor specificities. It was successfully applied to screen an E649 saturation mutagenesis library of Cellulomonas uda cellobiose phosphorylase (CP) for novel acceptor specificity. An E649C enzyme variant was found with novel acceptor specificity towards alkyl β-glucosides and phenyl β-glucoside. This is the first report of a CP enzyme variant with modified acceptor specificity.

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“…2). These results indicate that the phosphorolytic reaction of GalGlyNAcP Vv followed the sequential bi-bi mechanism, like the reactions of inverting phosphorylases, such as GalGlyNAcP from B. bifidum (5), GalGlyNAcP Bl (27), GalGlyNAcP Cp (27), maltose phosphorylase (39), cellobiose phosphorylase (15,17,28,34), chitobiose phosphorylase (12), and laminaribiose phosphorylase (18).…”

Section: Resultsmentioning

confidence: 88%

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

Nakajima

Kitaoka

2008

Appl Environ Microbiol

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A ␤-1,3-galactosyl-N-acetylhexosamine phosphorylase (GalGlyNAcP) homolog gene was cloned from Vibrio vulnificus CMCP6. In synthetic reactions, the recombinant enzyme acted only with GlcNAc and GalNAc as acceptors in the presence of ␣-D-galactose-1-phosphate as a donor to form lacto-N-biose I (LNB) (Gal␤1 3 3GlcNAc) and galacto-N-biose (GNB) (Gal␤1 3 3GalNAc), respectively. GlcNAc was a much better acceptor than GalNAc. The enzyme also phosphorolysed LNB faster than it phosphorolysed GNB, and the k cat /K m for LNB was approximately 60 times higher than the k cat /K m for GNB. This result indicated that the enzyme was remarkably different from GalGlyNAcP from Bifidobacterium longum, which has similar activities with LNB and GNB, and GalGlyNAcP from Clostridium perfringens, which is a GNB-specific enzyme. The enzyme is the first LNB-specific enzyme that has been found and was designated lacto-N-biose I phosphorylase. The discovery of an LNB-specific GalGlyNAcP resulted in recategorization of bifidobacterial GalGlyNAcPs as galacto-N-biose/ lacto-N-biose I phosphorylases.

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Characterization of a Cellobiose Phosphorylase from a Hyperthermophilic Eubacterium,Thermotoga maritimaMSB8

Cited by 42 publications

References 1 publication

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Kinetics and Relative Importance of Phosphorolytic and Hydrolytic Cleavage of Cellodextrins and Cellobiose in Cell Extracts of Clostridium thermocellum

Development and application of a screening assay for glycoside phosphorylases

Identification of Lacto-N-Biose I Phosphorylase fromVibrio vulnificusCMCP6

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