Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

Ntarima, Patricia; Nerinckx, Wim; Klarskov, Klaus; Devreese, Bart; Bhat, M. K.; Beeumen, Jozef Van; Claeyssens, Marc

doi:10.1042/0264-6021:3470865

Cited by 10 publications

(9 citation statements)

References 37 publications

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“…Oxirane-containing compounds have been used extensively as affinity labels of enzymes, and the subsequent characterization of the inactivated enzyme has provided much mechanistic insight (22–24). Ring opening, with the concomitant alkylation and inactivation, generally involves acid−base catalysis and proceeds by one of two mechanisms (25).…”

Section: Discussionmentioning

confidence: 99%

Inactivation of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue in Corynebacterium glutamicum, by (R)- and (S)-Oxirane-2-carboxylate: Analysis and Implications

Robertson

Johnson

et al. 2008

Biochemistry

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(R)- and (S)-oxirane-2-carboxylate were determined to be active site-directed irreversible inhibitors of the cis-3-chloroacrylic acid dehalogenase (cis-CaaD) homologue Cg10062 found in Corynebacterium glutamicum. Kinetic analysis indicates that the (R) enantiomer binds more tightly and is the more potent inhibitor, likely reflecting more favorable interactions with active site residues. Pro-1 is the sole site of covalent modification by the (R) and (S) enantiomers. Pro-1, Arg-70, Arg-73, and Glu-114, previously identified as catalytic residues in Cg10062, have also been implicated in the inactivation mechanism. Pro-1, Arg-70, and Arg-73 are essential residues for the process as indicated by the observation that the enzymes with the corresponding alanine mutations are not covalently modified by either enantiomer. The E114Q mutant slows covalent modification of Cg10062 but does not prevent it. The results are comparable to those found for the irreversible inactivation of cis-CaaD by (R)-oxirane-2-carboxylate with two important distinctions: the alkylation of cis-CaaD is stereospecific, and Glu-114 does not take part in the cis-CaaD inactivation mechanism. Cg10062 exhibits low-level cis-CaaD and trans-3-chloroacrylic acid dehalogenase (CaaD) activities, with the cis-CaaD activity predominating. Hence, the preference of Cg10062 for the cis isomer correlates with the observation that the (R) enantiomer is the more potent inactivator. Moreover, the factors responsible for the relaxed substrate specificity of Cg10062 may account for the stereoselective inactivation by the enantiomeric epoxides. Delineation of these factors would provide a more complete picture of the substrate specificity determinants for cis-CaaD. This study represents an important step toward this goal by setting the stage for a crystallographic analysis of inactivated Cg10062.

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Section: Discussionmentioning

confidence: 99%

Inactivation of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue in Corynebacterium glutamicum, by (R)- and (S)-Oxirane-2-carboxylate: Analysis and Implications

Robertson

Johnson

et al. 2008

Biochemistry

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“…Because we obtained similar results for activity and binding affinity when testing both of these mutant enzymes (results not shown), a single-site binding model was used to fit the data obtained for XBS_E172A_AAA. In the third variant, binding in the AS was blocked using the mechanism-based inhibitor 2,3-epoxypropyl β-D-xylopyranoside, which was covalently attached to the catalytic nucleophile of XBS (E78) [8]. The inactivation of the enzyme was performed analogously to what was described by Ntarima and coworkers [8].…”

Section: Hhmi Author Manuscriptmentioning

confidence: 99%

“…In the third variant, binding in the AS was blocked using the mechanism-based inhibitor 2,3-epoxypropyl β-D-xylopyranoside, which was covalently attached to the catalytic nucleophile of XBS (E78) [8]. The inactivation of the enzyme was performed analogously to what was described by Ntarima and coworkers [8]. After complete inactivation of the enzyme, as assessed by measurement of the residual activity on Xylazyme AX analogous to the previous description [6], the residual unbound inhibitor was washed out using Vivaspin 15R (Sartorius, Aubagne, France).…”

mentioning

confidence: 99%

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

Cuyvers¹,

Dornez²,

Hachem³

et al. 2012

Analytical Biochemistry

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Isothermal titration calorimetry and surface plasmon resonance were tested for their ability to study substrate binding to the active site (AS) and to the secondary binding site (SBS) of Bacillus subtilis xylanase A separately. To this end, three enzyme variants were compared. The first was a catalytically incompetent enzyme that allows substrate binding to both the AS and SBS. In the second enzyme, binding to the SBS was impaired by site-directed mutagenesis, whereas in the third enzyme, the AS was blocked using a covalent inhibitor. Both techniques were able to show that AS and SBS have a similar binding affinity. KeywordsIsothermal titration calorimetry; Surface plasmon resonance; Xylanase; Xylooligosaccharides; Mechanism-based inhibitor In various glycoside hydrolases, substrate molecules can bind not only to the active site (AS) 1 but also to remote sites on the surface of the catalytic module. These sites are referred to as secondary binding sites (SBSs) [1]. Several putative roles have been attributed to them, many of which are analogous to functions of carbohydrate-binding modules (CBMs) HHMI Author ManuscriptHHMI Author Manuscript HHMI Author Manuscript saccharides to the Bacillus circulans xylanase AS and SBS occurs independently, whereas binding of longer chains to both sites occurs cooperatively [4]. The binding affinity constants of short xylooligo-saccharides were similar for the AS and SBS, but the on-and off-rates of substrate binding were estimated to be at least 10-fold higher for the SBS [4]. In the study presented here, the potential of isothermal titration calorimetry (ITC) and SPR, two commonly used techniques quantifying binding, was explored for their ability to quantify the strength of substrate binding to the AS and SBS of the glycoside hydrolase family (GH)-11 Bacillus subtilis xylanase (XBS) separately. XBS differs only one residue from the B. circulans xylanase, and the SBSs of the two enzymes are identical [5]. The SBS plays an important role for XBS in binding and hydrolysis of polymeric substrates and in substrate targeting [6,7].In contrast to NMR-monitored titration, both ITC and SPR do not allow assessment of binding to the two sites individually in a direct way. Therefore, three enzyme variants were produced, and their purity was verified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and silver staining analogous to previous descriptions [6,7]. The first one, XBS_E172A, was a catalytically incompetent mutant that can still bind substrate to the AS and SBS [5]. A two-site binding model was used to fit the data obtained with XBS_E172A. In the second variant, XBS_E172A_AAA, catalytic activity was eliminated and binding to the SBS was also impaired by mutation of three important SBS residues: G56A, T183A, and W185A [6]. Previously, using NMR-monitored titration, Ludwiczek and coworkers [4] could not detect oligosaccharide binding to the SBS of a similar mutant enzyme in which also three important SBS residues were mutated to alanine. Because we...

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“…However, more data on other neutral and acidic endoxylanases are needed to verify this. Recently, a method was developed to experimentally determine whether an endoxylanase belongs to family 10 or 11 (272). This method is based on the irriversible inhibition of family 11 endoxylanases by epoxyl glycosides of D-xylose and xylooligosaccharides, whereas family 10 endoxylanases are unaffected (272).…”

Section: Degradation Of the Xylan Backbonementioning

confidence: 99%

AspergillusEnzymes Involved in Degradation of Plant Cell Wall Polysaccharides

Vries

Visser

2001

Microbiol Mol Biol Rev

885

585

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SUMMARY Degradation of plant cell wall polysaccharides is of major importance in the food and feed, beverage, textile, and paper and pulp industries, as well as in several other industrial production processes. Enzymatic degradation of these polymers has received attention for many years and is becoming a more and more attractive alternative to chemical and mechanical processes. Over the past 15 years, much progress has been made in elucidating the structural characteristics of these polysaccharides and in characterizing the enzymes involved in their degradation and the genes of biotechnologically relevant microorganisms encoding these enzymes. The members of the fungal genus Aspergillus are commonly used for the production of polysaccharide-degrading enzymes. This genus produces a wide spectrum of cell wall-degrading enzymes, allowing not only complete degradation of the polysaccharides but also tailored modifications by using specific enzymes purified from these fungi. This review summarizes our current knowledge of the cell wall polysaccharide-degrading enzymes from aspergilli and the genes by which they are encoded.

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Epoxyalkyl glycosides of d-xylose and xylo-oligosaccharides are active-site markers of xylanases from glycoside hydrolase family 11, not from family 10

Cited by 10 publications

References 37 publications

Inactivation of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue in Corynebacterium glutamicum, by (R)- and (S)-Oxirane-2-carboxylate: Analysis and Implications

Inactivation of Cg10062, a cis-3-Chloroacrylic Acid Dehalogenase Homologue in Corynebacterium glutamicum, by (R)- and (S)-Oxirane-2-carboxylate: Analysis and Implications

Isothermal titration calorimetry and surface plasmon resonance allow quantifying substrate binding to different binding sites of Bacillus subtilis xylanase

AspergillusEnzymes Involved in Degradation of Plant Cell Wall Polysaccharides

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