The Acid/Base Catalyst in the Exoglucanase/Xylanase from Cellulomonas fimi Is Glutamic Acid 127: Evidence from Detailed Kinetic Studies of Mutants

MacLeod, Alasdair M.; Lindhorst, Thisbe K.; Withers, Stephen G.; Warren, R. A. J.

doi:10.1021/bi00186a042

Cited by 153 publications

(134 citation statements)

References 23 publications

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“…This increase in activity is markedly high, with k cat values increasing by almost 10 3 -fold, approaching the rates of the wild type enzyme. Rate enhancements by azide were also observed for other acid-base mutants, although in these cases only up to a 3 ϫ 10 2 -fold increase was achieved (32,35). The presence of azide also affected the K m values, and these increased dramatically until leveling off (Fig.…”

Section: Fig 2 Brønsted Plots Of the Hydrolysis Of Aryl ␤-D-xylopyrmentioning

confidence: 76%

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Bravman

Belakhov

Solomon

et al. 2003

Journal of Biological Chemistry

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␤-D-Xylosidases (EC 3.2.1.37) are exo-type glycoside hydrolases that hydrolyze short xylooligosaccharides to xylose units. The enzymatic hydrolysis of the glycosidic bond involves two carboxylic acid residues, and their identification, together with the stereochemistry of the reaction, provides crucial information on the catalytic mechanism. Two catalytic mutants of a ␤-xylosidase from Geobacillus stearothermophilus T-6 were subjected to detailed kinetic analysis to verify their role in catalysis. The activity of the E335G mutant decreased ϳ10 6 -fold, and this activity was enhanced 10 3 -fold in the presence of external nucleophiles such as formate and azide, resulting in a xylosyl-azide product with an opposite anomeric configuration. These results are consistent with Glu 335 as the nucleophile in this retaining enzyme. The D495G mutant was subjected to detailed kinetic analysis using substrates bearing different leaving groups (pK a ). The mutant exhibited 10 3 -fold reduction in activity, and the Brønsted plot of log(k cat ) versus pK a revealed that deglycosylation is the rate-limiting step, indicating that this step was reduced by 10 3 -fold. The rates of the glycosylation step, as reflected by the specificity constant (k cat /K m ), were similar to those of the wild type enzyme for hydrolysis of substrates requiring little protonic assistance (low pK a ) but decreased 10 2 -fold for those that require strong acid catalysis (high pK a ). Furthermore, the pH dependence profile of the mutant enzyme revealed that acid catalysis is absent. Finally, the presence of azide significantly enhanced the mutant activity accompanied with the generation of a xylosyl-azide product with retained anomeric configuration. These results are consistent with Asp 495 acting as the acid-base in XynB2.

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Section: Fig 2 Brønsted Plots Of the Hydrolysis Of Aryl ␤-D-xylopyrmentioning

confidence: 76%

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Bravman

Belakhov

Solomon

et al. 2003

Journal of Biological Chemistry

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“…Cex and the papain-cleaved product, CexCD (residues 1-315), were expressed and purified by cellulose affinity chromatography, according to previously published protocols (34,35). CexCBD (residues 336 -443) was expressed directly using the plasmid pTug-KH 6 -IEGR-CBM2a in E. coli BL21 (DE3) cells (36,37).…”

Section: Expression and Purification Of Uniformlymentioning

confidence: 99%

“…The three cultures (totaling 4.5 liters) were grown in an incubator shaker at 30°C for 4 days, after which the S. lividans mycelium was removed by vacuum filtration through a Whatman glass microfiber filter. The supernatant, containing secreted glycosylated Cex, was incubated with CF-1 cellulose and purified by affinity chromatography, as described previously, yielding 37 mg of protein from 4.5 liters of isotopically enriched medium (34,35). The purified protein was found to be heterogeneously glycosylated by SDS-PAGE and matrix-assisted laser desorption ionization-time of flight mass spectrometry analyses.…”

Section: Expression and Purification Of Uniformlymentioning

confidence: 99%

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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“…All experiments used the catalytic domain of Cex and its mutants, expressed and purified according to methods°adapted°from°earlier°procedures° [21][22][23].°Details°are given in the Appendix. The final stock solution concentrations of all the proteins were about 1-4 mg/mL.…”

Section: Protein Expression and Purificationmentioning

confidence: 99%

Gas phase noncovalent protein complexes that retain solution binding properties: Binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi

Tešić

Wicki

Poon

et al. 2007

J. Am. Soc. Mass Spectrom.

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Tandem mass spectrometry has been used to compare gas-phase and solution binding of three small-molecule inhibitors to the wild type and three mutant forms of the catalytic domain of Cex, an enzyme that hydrolyses xylan and xylo-oligosaccharides. The inhibitors, xylobiosyldeoxynojirimycin, xylobiosyl-isofagomine lactam, and xylobiosyl-isofagomine consist of a common distal xylose linked to different proximal aza-sugars. The three mutant forms of the enzyme contain the substitutions Asn44Ala, Gln87Met, and Gln87Tyr that alter the binding interactions between Cex and the distal sugar of each inhibitor. An electrospray ionization (ESI) triple quadrupole MS/MS system is used to measure the internal energies, ⌬E int , that must be added to gas-phase ions to cause dissociation of the noncovalent enzyme-inhibitor complexes. Collision cross sections of ions of the apo-enzyme and enzyme-inhibitor complexes, which are required for the calculations of ⌬E int , have also been measured. The results show that, in the gas phase, enzyme-inhibitor complexes have more compact, folded conformations than the corresponding apo-enzyme ions. With the mutant enzymes, the effects of substituting a single residue can be detected. The energies required to dissociate the gas-phase complexes follow the same trend as the values of ⌬G 0 for dissociation of the complexes in solution. This trend is observed both with different inhibitors, which probe binding to the proximal sugar, and with mutants of Cex, which probe binding to the distal sugar. Thus the gas-phase complexes appear to retain much of their solution binding characteristics. (J Am Soc Mass Spectrom 2007, 18, 64 -73)

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The Acid/Base Catalyst in the Exoglucanase/Xylanase from Cellulomonas fimi Is Glutamic Acid 127: Evidence from Detailed Kinetic Studies of Mutants

Cited by 153 publications

References 23 publications

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

Identification of the Catalytic Residues in Family 52 Glycoside Hydrolase, a β-Xylosidase from Geobacillus stearothermophilus T-6

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

Gas phase noncovalent protein complexes that retain solution binding properties: Binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi

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