Purification and mechanistic properties of an extracellular α-<scp>l</scp>-arabinofuranosidase from <i>Monilinia fructigena</i>

Kelly, Mark; Sinnott, Michael L.; Herrchen, Monika

doi:10.1042/bj2450843

Cited by 18 publications

(13 citation statements)

References 33 publications

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“…213-217 mC (dec.)] [25]q gave a "$C NMR spectrum in accord with that reported previously [25]. The amine was converted into the triazene by reaction with p-nitrobenzene diazonium tetrafluoroborate in buffered aqueous solution [26].…”

Section: Synthetic Substrates Inhibitors and Inactivatorssupporting

confidence: 78%

Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

1999

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The main alpha-galactosidase was purified to homogeneity, in 30% yield, from a solid culture of Phanerochaete chrysosporium on 1 part wheat bran/2 parts thermomechanical softwood pulp. It is a glycosylated tetramer of 50 kDa peptide chains, which gives the N-terminal sequence ADNGLAITPQMG(?W)NT(?W)NHFG(?W)DIS(?W)DTI. It is remarkably stable, with crude extracts losing no activity over 3 h at 80 degrees C, and the purified enzyme retaining its activity over several months at 4 degrees C. The kinetics of hydrolysis at 25 degrees C of various substrates by this retaining enzyme were measured, absolute parameters being obtained by active-site titration with 2',4',6'-trinitrophenyl 2-deoxy-2, 2-difluoro-alpha-D-galactopyranoside. The variation of kcat/Km for 1-naphthyl-alpha-D-galactopyranoside with pH is bell-shaped, with pK1=1.91 and pK2=5.54. The alphaD(V/K) value for p-nitrophenyl-alpha-D-glucopyranoside is 1.031+/-0.007 at the optimal pH of 3.75 and 1.114+/-0.006 at pH7.00, indicating masking of the intrinsic effect at optimal pH. There is no alpha-2H effect on binding galactose [alphaD(Ki)=0.994+/-0.013]. The enzyme hydrolyses p-nitrophenyl beta-L-arabinopyranoside approximately 510 times slower than the galactoside, but has no detectable activity on the alpha-D-glucopyranoside or alpha-D-mannopyranoside. Hydrolysis of alpha-galactosides with poor leaving groups is Michaelian, but that of substrates with good leaving groups exhibits pronounced apparent substrate inhibition, with Kis values similar to Km values. We attribute this to the binding of the second substrate molecule to a beta-galactopyranosyl-enzyme intermediate, forming an E.betaGal. alphaGalX complex which turns over slowly, if at all. 1-Fluoro-alpha-D-galactopyranosyl fluoride, unlike alpha-D-galactopyranosyl fluoride, is a Michaelian substrate, indicating that the effect of 1-fluorine substitution is greater on the first than on the second step of the enzyme reaction.

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Section: Synthetic Substrates Inhibitors and Inactivatorssupporting

confidence: 78%

Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

1999

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“…Peak I showed optimal activity for pNP-a-L-arabinofuranoside (pNPAf) rather than for pNPX. This characteristic has been already observed for a-L-arabinofuranosidases isolated from other sources (Kelly et al, 1987;Beylot et al, 2001). Peak II showed an optimal efficiency with the natural substrate xylobiose suggesting that this enzyme is more specifically a b-D-xylosidase.…”

Section: Substrate Specificities Of the Purified Enzymessupporting

confidence: 75%

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

et al. 2004

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This work describes the purification and characterization of enzymes that exhibit b-D-xylosidase activity in stem tissues of Arabidopsis. This is the first detailed investigation that concerns the characterization of catalytic properties and sequence identity of enzymes with b-D-xylosidase activities in a dicotyledonous plant. Three different enzymes, ARAf, XYL4, and XYL1 with apparent molecular masses of 75, 67, and 64 kD, respectively, were purified to homogeneity. ARAf was identified as a putative a-L-arabinofuranosidase, and XYL4 and XYL1 as putative b-D-xylosidases using matrix-assisted laser-desorption ionization time of flight. ARAf belongs to family 51 and XYL4 and XYL1 to family 3 of glycoside hydrolases. ARAf and XYL1 have highest specificity for p-nitrophenyl-a-L-arabinofuranoside and XYL4 for p-nitrophenyl-b-D-xylopyranoside and natural substrates such as xylobiose and xylotetraose. XYL4 was shown to release mainly D-Xyl from oat spelt xylan, rye arabinoxylan, wheat arabinoxylan, and oligoarabinoxylans. ARAf and XYL1 can also release D-Xyl from these substrates but less efficiently than XYL4. Moreover, they can also release L-Ara from arabinoxylans and arabinan. Overall, the results indicate that XYL4 possesses enzymatic specificity characteristic for a b-D-xylosidase, while ARAf and XYL1 act as bifunctional a-L-arabinofuranosidase/b-D-xylosidases. Analysis of the activity of these three enzymes in stem tissues at different stages of development has shown that young stems possess the highest activities for all three enzymes in comparison to the activities of the enzymes present in stems at older stages of development. High enzyme activities are most likely related to the necessary modifications of cell wall structure occurring during plant growth. Plant cells are surrounded by an extracellular matrix known as the cell wall. The regulation of cell wall morphology and composition is important for the determination of cell size and shape, cellular interactions with the environment, mechanical resistance, and defense against pathogen attacks (Reiter, 2002). The cell wall is also involved in plant growth and development (Stolle-Smits et al., 1999;Obel et al., 2002). In addition, although cell walls consist mainly of polysaccharides broadly classified as celluloses, hemicelluloses, and pectins, their constitution varies, not only from plant to plant, but also in different tissues of the same plant (Heredia et al., 1995;Cosgrove, 1997;Popper and Fry, 2003). A coordinated series of biochemical processes occur during plant development resulting in the biosynthesis and degradation of cell wall components. Consequently, numerous enzymes must be implicated in these processes (Heredia et al., 1995;Cosgrove, 1997;Stolle-Smits et al., 1999;Obel et al., 2002;Reiter, 2002;Popper and Fry, 2003). However, to date, little information is available concerning such enzymes, their mode of action, and their physiological role. Increased research interest has focused on enzymes involved in the metabolism of hemicelluloses in ...

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“…The k cat /K m values reflect the first irreversible step, and, therefore, for retaining glycoside hydrolases the dependence of these values on the pK a of the phenol leaving group provides information about the glycosylation step (28,35). To date, this kind of detailed kinetic analysis with ␣-L-arabinofuranosidases was applied only for the enzyme from Monilinia fructigena (36). Because the amino acid sequence of this enzyme (and hence its GH classification) is not known, the results of that study could not be extended to other ␣-L-arabinofuranosidases.…”

Section: Resultsmentioning

confidence: 99%

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

Shallom¹,

Belakhov²,

Solomon³

et al. 2002

Journal of Biological Chemistry

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␣-L-Arabinofuranosidases cleave the L-arabinofuranoside side chains of different hemicelluloses and are key enzymes in the complete degradation of the plant cell wall. The ␣-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-␣-Larabinofuranosides with various leaving groups were synthesized and used to verify the catalytic mechanism and catalytic residues of the enzyme. The steady-state constants and the resulting Brønsted plots for the E175A mutant are consistent with the role of Glu-175 as the acid-base catalytic residue. The proposed nucleophile residue, Glu-294, was replaced to Ala by a double-base pairs substitution. The resulting E294A mutant, with 4-nitrophenyl ␣-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher K m value compared with the wild type enzyme. Sodium azide accelerated by more than 40-fold the rate of the hydrolysis of 2 ,4 ,6 -trichlorophenyl ␣-Larabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as ␤-L-arabinofuranosyl-azide by 1 H NMR, 13 C NMR, mass spectrometry, and Fourier transform infrared analysis. The anomeric configuration of this product supports the assignment of Glu-294 as the catalytic nucleophile residue of the ␣-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.␣-L-Arabinofuranosidases (EC 3.2.1.55) are hemicellulases that cleave the glycosidic bond between L-arabinofuranosides side chains and different oligosaccharides. These enzymes are part of an array of glycoside hydrolases responsible for the degradation of hemicelluloses such as arabinoxylan, arabinogalactan, and L-arabinan (1-3). The L-arabinofuranoside substitutions on xylans can strongly inhibit the action of endoxylanases and ␤-xylosidases, thus preventing the complete degradation of the polymer to its basic xylose units (4, 5). In many cases, microorganisms that utilize hemicelluloses possess ␣-L-arabinofuranosidases with various substrate specificities and biochemical properties (6). To date, there are more than 110 sequences of different ␣-L-arabinofuranosidases, which are classified, based on sequence homology, into four glycoside hydrolase families (GHs) 1 : GH43, GH51, GH54, and GH62 (7,8). Hemicellulases, together with cellulases, have a key role in the carbon cycle in nature, because they are responsible for the complete degradation of the plant biomass to soluble saccharides. These in turn can be used as carbon or energy sources for microorganisms and higher animals. Hemicellulases have attracted much attention in recent years because of their potential industrial use in biobleaching of paper pulp, bioconversion of lignocellulose material to fermentative products, improvement of animal feedstock digestibility, and organic synthesis (6, 9 -11).The glycosidic bond is one of the most stable bonds in...

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Purification and mechanistic properties of an extracellular α-l-arabinofuranosidase from Monilinia fructigena

Cited by 18 publications

References 33 publications

Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

Lignocellulose degradation by Phanerochaete chrysosporium: purification and characterization of the main α-galactosidase

Purification and Characterization of Enzymes Exhibiting β-d-Xylosidase Activities in Stem Tissues of Arabidopsis

Detailed Kinetic Analysis and Identification of the Nucleophile in α-l-Arabinofuranosidase from Geobacillus stearothermophilus T-6, a Family 51 Glycoside Hydrolase

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