Biochim Biophys Acta Gen Subj 2019 Jun 01;18636:1070-1078. doi: 10.1016/j.bbagen.2019.03.020.

Kinetics and regioselectivity of three GH62 α-L-arabinofuranosidases from plant pathogenic fungi.

Abstract
BACKGOUND: Xylan is the second most abundant plant cell wall polysaccharide after cellulose with α-L-arabinofuranose (L-Araf) as one of the major side substituents. Capacity to degrade xylan is characteristic of many plant pathogens; and corresponding enzymes that debranch arabinoxylan provide tools to tailor xylan functionality or permit its full hydrolysis. METHOD: Three GH62_2 family α-arabinofuranosidases (Abfs) from plant pathogenic fungi, NhaAbf62A from Nectria haematococca, SreAbf62A from Sporisorium reilianum and GzeAbf62A from Gibberella zeae, were recombinantly produced in Escherichia coli. Their biochemical properties and substrate specificities were characterized in detail. Particularly with 1H NMR, the regioselectivity and debranching preference of the three Abfs were directly compared. RESULTS: The activities of selected Abfs towards arabinoxylan were all optimal at pH 6.5. Their preferred substrates were wheat arabinoxylan, followed by soluble oat spelt xylan. The Abfs displayed selectivity towards either α-(1 → 2) or α-(1 → 3)-L- Araf mono-substituents in arabinoxylan. Specifically, SreAbf62A and GzeAbf62A removed m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substituents with a similar rates, whereas NhaAbf62A released m-α-(1 → 3)-L-Araf 1.9 times faster than m-α-(1 → 2)-L-Araf. MAJOR CONCLUSIONS: Building upon the known selectivity of GH62 family α-arabinofuranosidases towards L-Araf mono-substituents in xylans, the current study uncovers enzyme-dependent preferences towards m-α-(1 → 3)-L-Araf and m-α-(1 → 2)-L-Araf substitutions. Comparative sequence-structure analyses of Abfs identified an arginine residue in the xylose binding +2R subsite that was correlated to the observed enzyme-dependent L-Araf debranching preferences. GENERAL SIGNIFICANCE: This study expands the limited pool of characterized GH62 Abfs particularly those from plant pathogenic fungi, and provides biochemical details and methodology to evaluate regioselectivity within this glycoside hydrolase family.

PubMed ID: 30936018
Article link: Biochim Biophys Acta Gen Subj


Genes referenced: LOC115919910 LOC593316