Detailed kinetic analysis and identification of the nucleophile in α-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase

Dalia Shallom, Valery Belakhov, Dmitry Solomon, Gil Shoham, Timor Baasov, Yuval Shoham*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

91 Scopus citations

Abstract

α-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 α-L-arabinofuranosidase from Geobacillus stearothermophilus T-6, a family 51 glycoside hydrolase, was subjected to a detailed mechanistic study. Aryl-α-L-arabinofuranosides 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 α-L-arabinofuranoside as the substrate, exhibited eight orders of magnitude lower activity and a 10-fold higher Km 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 α-L-arabinofuranoside by the E294A mutant. The glycosylazide product formed during this reaction was isolated and characterized as β-L-arabinofuranosyl-azide by 1H NMR, 13C 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 α-L-arabinofuranosidase T-6 and allows for the first time the unequivocal identification of this residue in glycoside hydrolases family 51.

Original languageAmerican English
Pages (from-to)43667-43673
Number of pages7
JournalJournal of Biological Chemistry
Volume277
Issue number46
DOIs
StatePublished - 15 Nov 2002

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