Crystal Structure of an Exo-1,5-α-l-arabinofuranosidase from Streptomyces avermitilis Provides Insights into the Mechanism of Substrate Discrimination between Exo- and Endo-type Enzymes in Glycoside Hydrolase Family 43*

Abstract

Exo-1,5-␣-L-arabinofuranosidases belonging to glycoside hydrolase family 43 have strict substrate specificity. These enzymes hydrolyze only the ␣-1,5-linkages of linear arabinan and arabino-oligosaccharides in an exo-acting manner. The enzyme from Streptomyces avermitilis contains a core catalytic domain belonging to glycoside hydrolase family 43 and a C-terminal arabinan binding module belonging to carbohydrate binding module family 42. We determined the crystal structure of intact exo-1,5-␣-L-arabinofuranosidase. The catalytic module is composed of a 5-bladed ␤-propeller topologically identical to the other family 43 enzymes. The arabinan binding module had three similar subdomains assembled against one another around a pseudo-3-fold axis, forming a ␤-trefoil-fold. A sugar complex structure with ␣-1,5-L-arabinofuranotriose revealed three subsites in the catalytic domain, and a sugar complex structure with ␣-L-arabinofuranosyl azide revealed three arabinose-binding sites in the carbohydrate binding module. A mutagenesis study revealed that substrate specificity was regulated by residues Asn-159, Tyr-192, and Leu-289 located at the aglycon side of the substrate-binding pocket. The exo-acting manner of the enzyme was attributed to the strict pocket structure of subsite ؊1, formed by the flexible loop region Tyr-281-Arg-294 and the side chain of Tyr-40, which occupied the positions corresponding to the catalytic glycon cleft of GH43 endo-acting enzymes.L-Arabinose residues are widely distributed in plant cell walls, where they are present in polymers such as arabinans, arabinoxylans, arabinogalactans, and arabinogalactan proteins (1). Research on plant cell walls is becoming a necessity because worldwide attention has now focused on bioethanol production to combat global warming and to improve global energy security. Because of competition between food and fuel, lignocellulose is expected to be used as a material for fuel ethanol production in the future. Generally, lignocellulose contains cellulose, which makes up ϳ40% of the total amount of cell wall components, together with ϳ20% hemicellulose, which is mainly composed of pentoses such as xylose and arabinose (2). Hemicelluloses often become bad factors in bioethanol production because the efficiency of ethanol conversion from pentoses is significantly lower than that from hexoses (3, 4).In contrast, L-arabinose is used as a functional sugar in the food industry. This sugar has a sweet taste and selectively inhibits intestinal sucrase activity in a noncompetitive manner and consequently suppresses plasma glucose increase due to sucrose ingestion (5-7). Therefore, L-arabinose may also be useful in preventing excess sucrose utilization.Because the structure of L-arabinose-containing polysaccharides is highly variable and complex, a wide variety of ␣-L-arabinofuranosidases (EC 3.2.1.55) that have various substrate specificities are necessary for the hydrolysis of such polysaccharides and for the production of L-arabinose. We have previously purified some ␣-L-arabi...

The Structure and Function of an Arabinan-specific α-1,2-Arabinofuranosidase Identified from Screening the Activities of Bacterial GH43 Glycoside Hydrolases

“…It is difficult, therefore, to understand how these enzymes distinguish between an Araf and Xylp at the critical Ϫ1 subsite because arabinofuranosidases that hydrolyze 4NP-Araf are not active on 4NP-Xylp. Thus, Asp 41 , Glu 215 , and Asp 168 , which function as the catalytic base, catalytic acid, and modulator of the pK a of the catalytic acid, respectively, are conserved in GH43 enzymes (8,10,11,31,32). Consistent with their catalytic function, alanine substitution of these three residues inactivates the arabinofuranosidase ( Enzyme concentrations varied from 5 nM to 10 M. Reactions were carried out at 25°C in 50 mM sodium phosphate buffer, pH 7.0.…”

“…CjAbf43A displays a five-bladed ␤-propeller fold (Fig. 4), typical of GH43 enzymes (8,10,11,32,38). It has a cylindrical shape with a diameter and height of 35 Å.…”

“…japonicus-Biochemical analysis of the two exo-␣-1,5-arabinofuranosidases, CJA_3012 and CJA_0806 ( Table 2), showed that both enzymes displayed the highest catalytic activity against linear arabinan and arabinooligosaccharides, releasing exclusively arabinose, probably from the non-reducing end (all exo-acting GH43 enzymes attack the non-reducing end of polysaccharides (8,11,31,32)). The two enzymes released only very small amounts of arabinose from sugar beet, reflecting the limited concentration of terminal undecorated ␣-1,5-arabinofuranose units in this highly branched polysaccharide (data not shown).…”

“…The N-and C-terminal blades, however, are in close association, and stability is provided by strong hydrogen bonds between Asp 78 in blade 1 and several residues in blade 5. CjAbf43A lacks a C-terminal ␤-jelly roll domain seen in several exo-acting GH43 enzymes (8,10,11,31).…”

Introducing endo-xylanase activity into an exo-acting arabinofuranosidase that targets side chains

“…The active site of HiAXHd3, located in the center of the xylotriose binding cleft, contains a constellation of carboxylate residues, Asp43, Asp167, and Glu216, which are invariant within GH43. The equivalent residues have been shown to comprise the catalytic amino acids in other members of the family (7,(11)(12)(13) (Fig. S3).…”