Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi

MacLeod, Alasdair M.; Gilkes, Neil R.; Escote-Carlson, Loida; Antony, Robert J.; Warren, James W.; Kilburn, Douglas G.; Miller, Robert C.

doi:10.1016/0378-1119(92)90173-m

Cited by 22 publications

(21 citation statements)

References 21 publications

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“…However, it remains to be established whether or not this alters the activity of Cex toward its natural substrates. To date, the best documented role of glycosylation is to enhance the viability of Cex in an extracellular environment by providing protection for the proteolytically vulnerable PT linker (31). In addition, the presence of glycans has been reported to marginally increase the relative affinity of a Cex fragment, comprising only of the PT linker plus CexCBD, relative to its nonglycosylated counterpart for crystalline cellulose, possibly through direct sugar-sugar interactions (30).…”

Section: Glycosylation Partially Dampens the Fast Time Scale Motions mentioning

confidence: 99%

“…Furthermore, this PT linker is O-glycosylated with ␣-D-mannose and ␣-D-galactose, such that ϳ24 mol of hexose are found per mole of Cex secreted by C. fimi (30). Early studies demonstrated that this modification leads to resistance of the Cex linker region against proteolytic degradation but that glycosylation has no significant effect upon kinetic parameters for hydrolysis of polymeric substrates (31). Without a method for the overexpression of Cex in C. fimi, only very small quantities of the endogenously glycosylated protein can be obtained.…”

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confidence: 99%

“…Without a method for the overexpression of Cex in C. fimi, only very small quantities of the endogenously glycosylated protein can be obtained. Fortunately, using Streptomyces lividans, Cex can be expressed and glycosylated in milligram amounts, as required for NMR spectroscopic characterization (30,31). Glycosylated Cex from S. lividans is reported to contain ϳ17 mol of mannose, with traces of galactose, per mole of protein (30,31).…”

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confidence: 99%

“…Fortunately, using Streptomyces lividans, Cex can be expressed and glycosylated in milligram amounts, as required for NMR spectroscopic characterization (30,31). Glycosylated Cex from S. lividans is reported to contain ϳ17 mol of mannose, with traces of galactose, per mole of protein (30,31). These differences are minor because samples of O-glycosylated Cex from C. fimi and S. lividans have very similar kinetic properties and are both resistant toward proteolytic degradation (31,32).…”

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confidence: 99%

“…Glycosylated Cex from S. lividans is reported to contain ϳ17 mol of mannose, with traces of galactose, per mole of protein (30,31). These differences are minor because samples of O-glycosylated Cex from C. fimi and S. lividans have very similar kinetic properties and are both resistant toward proteolytic degradation (31,32). Based on chemical shift, 15 N relaxation, and amide residual dipolar coupling (RDC) measurements, we demonstrate that the catalytic and cellulose-binding domains are physically independent and joined by a PT linker that is conformationally dynamic on the ns-to-ps time scale.…”

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confidence: 99%

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Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Poon

Withers

McIntosh

2007

Journal of Biological Chemistry

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The modular xylanase Cex (or CfXyn10A) from Cellulomonas fimi consists of an N-terminal catalytic domain and a C-terminal cellulose-binding domain, joined by a glycosylated proline-threonine (PT) linker. To characterize the conformation and dynamics of the Cex linker and the consequences of its modification, we have used NMR spectroscopy to study full-length Cex in its nonglycosylated (ϳ47 kDa) and glycosylated (ϳ51 kDa) forms. The PT linker lacks any predominant structure in either form as indicated by random coil amide chemical shifts. Furthermore, heteronuclear 1 H-15 N nuclear Overhauser effect relaxation measurements demonstrate that the linker is flexible on the ns-to-ps time scale and that glycosylation partially dampens this flexibility. The catalytic and cellulose-binding domains also exhibit identical amide chemical shifts whether in isolation or in the context of either unmodified or glycosylated full-length Cex. Therefore, there are no noncovalent interactions between the two domains of Cex or between either domain and the linker. This conclusion is supported by the distinct 15 N relaxation properties of the two domains, as well as their differential alignment within a magnetic field by Pf1 phage particles. These data demonstrate that the PT linker is a flexible tether, joining the structurally independent catalytic and cellulosebinding domains of Cex in an ensemble of conformations; however, more extended forms may predominate because of restrictions imparted by the alternating proline residues. This supports the postulate that the binding-domain anchors Cex to the surface of cellulose, whereas the linker provides flexibility for the catalytic domain to hydrolyze nearby hemicellulose (xylan) chains.Cellulolytic organisms produce a battery of endo-and exoglucanases necessary for the hydrolysis of cellulose and hemicellulose. These glycoside hydrolases are typically modular, consisting of conserved catalytic and carbohydrate-binding domains (or modules), as well as possible ancillary domains, joined by variable linker sequences (1-3). In general, the constituent domains of glycoside hydrolases are structurally independent and exhibit some aspects of their respective functions when separated (4 -6). Thus, binding modules appear to facilitate catalysis by targeting and maintaining the proximity of the catalytic domain toward substrates within complex macromolecular systems, such as the plant cell wall, as well as through possible disruptive effects on the structures of the polysaccharides within these systems (7,8).The synergistic activity of the catalytic and carbohydratebinding domains in a glycoside hydrolase requires that they be covalently tethered to one another via a linker sequence of the appropriate length and/or flexibility. The functional importance of these interdomain linkers, which can range from only a few to over a hundred residues and are often rich in proline and hydroxyamino acids (1), has been established largely through deletion studies (9 -12). However, the physical properties of g...

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Section: Glycosylation Partially Dampens the Fast Time Scale Motions mentioning

confidence: 99%

mentioning

confidence: 99%

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confidence: 99%

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confidence: 99%

mentioning

confidence: 99%

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Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Poon

Withers

McIntosh

2007

Journal of Biological Chemistry

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Cloning and characterization of the first actinomycete β‐propeller phytase from Streptomyces sp. US42

Boukhris

Farhat-Khemakhem

Bouchaala

et al. 2016

J. Basic Microbiol.

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A gene encoding an extracellular phytase was cloned for the first time from an Actinomycete, Streptomyces sp. US42 and sequenced. The sequence of this gene revealed an encoded polypeptide (PHY US42) exhibiting one and six residues difference with the putative phytases of Streptomyces lividans TK24 and Streptomyces coelicolor A3(2), respectively. The molecular modeling of PHY US42 indicated that this phytase belongs to the group of β-propeller phytases that are usually calcium-dependent. PHY US42 was purified and characterized. Its activity was calcium-dependent and maximal at pH 7 and 65 °C. The enzyme was perfectly stable at pH ranging from 5 to 10 and its thermostability was greatly enhanced in the presence of calcium. Indeed, PHY US42 maintained 80% of activity after 10 min of incubation at 75 °C in the presence of 5 mM CaCl . PHY US42 was also found to exhibit high stability after incubation at 37 °C for 1 h in the presence of bovine bile and digestive proteases like of pepsin, trypsin, and chymotrypsin. Considering its biochemical properties, PHY US42 could be used as feed additive in combination with an acid phytase for monogastric animals.

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Elicitation of lignin peroxidase inStreptomyces lividans

Yee

Wood

1996

Appl Biochem Biotechnol

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Streptomyces lividans glycosylates an exoglucanase (Cex) from Cellulomonas fimi

Cited by 22 publications

References 21 publications

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Direct Demonstration of the Flexibility of the Glycosylated Proline-Threonine Linker in the Cellulomonas fimi Xylanase Cex through NMR Spectroscopic Analysis

Cloning and characterization of the first actinomycete β‐propeller phytase from Streptomyces sp. US42

Elicitation of lignin peroxidase inStreptomyces lividans

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