Probing the catalytically essential residues of the α-l-arabinofuranosidase from Thermobacillus xylanilyticus

Abstract

The alpha-L-arabinofuranosidase D3 from Thermobacillus xylanilyticus is an arabinoxylan-debranching enzyme which belongs to family 51 of the glycosyl hydrolase classification. Previous studies have indicated that members of this family are retaining enzymes and may form part of the 4/7 superfamily of glycosyl hydrolases. To investigate the active site of alpha-L-arabinofuranosidase D3, we have used sequence alignment, site-directed mutagenesis and kinetic analyses. Likewise, we have shown that Glu(28), Glu(176) and Glu(298) are important for catalytic activity. Kinetic data obtained for the mutant Glu(176)-->Gln, combined with the results of chemical rescue using the mutant Glu(176)-->Ala, have shown that Glu(176) is the acid-base residue. Moreover, NMR analysis of the arabinosyl-azide adduct, which was produced by chemical rescue of the mutant Glu(176)-->Ala, indicated that alpha-L-arabinofuranosidase D3 hydrolyses glycosidic bonds with retention of the anomeric configuration. The results of similar chemical rescue studies using other mutant enzymes suggest that Glu(298) might be the catalytic nucleophile and that Glu(28) is a third member of a catalytic triad which may be responsible for modulating the ionization state of the acid-base and implicated in substrate fixation. Overall, these findings support the hypothesis that alpha-L-arabinofuranosidase D3 belongs to the 4/7 superfamily and provide the first experimental evidence concerning the catalytic apparatus of a family 51 arabinofuranosidase.

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

“…The vast decline in the k cat /K m values of the E175A mutant as the pK a of the phenol leaving group increases indicates that, for substrates that require strong acid assistance (such as 3-NPAF), the first step is much slower than for those substrates that need less acid assistance (such as 2,5-DNPAF). This kinetic behavior displayed by the E175A mutant compared with the wild type AbfA T-6 completely agrees with previous predictions (17)(18)(19)(20) and identifies E175 (and its homologous residues in the other arabinofuranosidases genes) as the general acid-base catalytic residues of GH51.…”

“…These results may imply that the formation of the transition state in the active site of the mutant enzyme is governed more by the structure of the aglycon, rather than by its reactivity. In two other GH51 ␣-L-arabinofuranosidases, from Pseudomonas cellulasa and from Thermobacillus xylanilyticus, the replacement of the corresponding residues had also resulted in very low activities (17,18). Chemical Rescue of the Inactive E294A Mutant-Although the substantial decrease in the catalytic activity of the mutant enzyme lacking the putative nucleophilic residue is consistent with its suggested role, the identification of the catalytic residues could not be based on the reduction in activity alone.…”

“…Both catalytic mechanisms are found in the different ␣-L-arabinofuranosidases families: GH51 and GH54 were shown to cleave the glycosidic bond with a retention of the anomeric configuration (15); GH43, which includes ␣-L-arabinofuranosidases as well as ␤-D-xylosidases, was shown to work via the inverting mechanism (15), whereas the stereochemistry of GH62 is not yet characterized. Currently there are 32 sequences of ␣-L-arabinofuranosidases classified as GH51 glycoside hydrolases, 2 and, although much work was done on the substrate specificities of these enzymes, there are only a few studies concerning the biochemical mechanism and catalytic properties of this family (17)(18)(19). Based on sequence similarities, GH51 is part of the GH-A superfamily (20), suggesting that the active site of the enzymes of this family is located at the C-terminal end of a (␤/␣) 8 fold.…”

Increasing the transglycosylation activity of α‐galactosidase from Bifidobacterium adolescentis DSM 20083 by site‐directed mutagenesis

“…Catalytic triads were also found in different types of enzymes (Amaya et al, 2003;Debeche et al, 2002;Schubot et al, 2001;Shaw et al, 2002). The residues D380 and D381 probably play a key role in substrate binding, through H-bonds between both Asp and the substrate (Brzozowski and Davies, 1997;Chen et al, 1995;Debeche et al, 2002;Namchuk and Withers, 1995), and may also play a role in influencing the ionization status of the catalytic residues (Brzozowski and Davies, 1997;Chen et al, 1995;Debeche et al, 2002).…”

Identification of a Novel α-Galactosidase from the Hyperthermophilic ArchaeonSulfolobus solfataricus

“…Unfortunately, reactivation experiments with external nucleophiles such as azide and formate failed to restore the activity of either the nucleophile or the A/B mutant enzymes. While ␤-glycosidases can sometimes be reactivated up to wild-type levels (37), for unknown reasons this strategy seems to be far less efficient for retaining ␣-glycosidases (6,9).…”