Barley arabinoxylan arabinofuranohydrolases: purification, characterization and determination of primary structures from cDNA clones

A family 51 arabinoxylan arabinofuranohydrolase, designated AXAH-I, has bee n purified from extracts of ri-day-old barley (Hordeum vulgare L.) seedling s by fractional precipitation with (NH,),SO, and ion-exchange chromatograph y, The enzyme has an apparent molecular mass of 65 kDa and releases L-arabi nose from cereal cell wall arabinoxylans with a pH optimum of 4.3, a cataly tic rate constant (k(cat)) of 6.9 s(-1) and a catalytic efficiency factor ( k(cat)/K-m) of 0.76 (ml . s(-1) . mg(-1)). Whereas the hydrolysis of alpha -L-arabinofuranosyl residues linked to C(O)3 of backbone (1-->4)-beta -xylo syl residues proceeds at the fastest rate, alpha -L-arabinofuranosyl residu es on doubly substituted xylosyl residues are also hydrolysed, at lower rat es. A near full-length cDNA encoding barley AXAH-I indicates that the matur e enzyme consists of 626 amino acid residues and has a calculated pi of 4.8 . A second cDNA, which is 81% identical with that encoding AXAH-I, encodes another barley AXAH, which has been designated AXAH-II. The barley AXAHs ar e likely to have key roles in wall metabolism in cereals and other members of the Poaceae. Thus the enzymes could participate in the modification of t he fine structure of arabinoxylan during wall deposition, maturation or exp ansion, or in wall turnover and the hydrolysis of arabinoxylans in germinat ed grain.