Exchanging a single amino acid residue generates or weakens a +2 cellooligosaccharide binding subsite in rice β-glucosidases

Os3BGlu6, Os3BGlu7, and Os4BGlu12 are rice glycoside hydrolase family 1 β-glucosidases, the structures of which have been solved by X-ray crystallography. In complex structures, Os3BGlu7 residue Asn245 hydrogen bonds to the second sugar in the +1 subsite for laminaribiose and the third sugar in the +2 subsite for cellotetraose and cellopentaose. The corresponding Os3BGlu6 residue, Met251, appears to block the binding of cellooligosaccharides at the +2 subsite, whereas His252 in this position in Os4BGlu12 could hydrogen bond to oligosaccharides. Mutation of Os3BGlu6 Met251 to Asn resulted in a 15-fold increased kcat/Km value for hydrolysis of laminaribiose compared to wild type Os3BGlu6 and 9 to 24-fold increases for cellooligosaccharides with degrees of polymerization (DP) of 2–5. On the other hand, mutation of Os3BGlu7 Asn245 to Met decreased the kcat/Km of hydrolysis by 6.5-fold for laminaribiose and 17 to 30-fold for cellooligosaccharides with DP >2, while mutation of Os4BGlu12 His252 to Met decreased the corresponding kcat/Km values 2 to 6-fold.

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► Os3BGlu7 Asn245 binds the cellooligosaccharide glucosyl residue in the +2 subsite.
► The corresponding Os3BGlu6 Met251 appears to block this residue.
► Mutation of Os3BGlu6 Met241 to Asn increased oligosaccharide kcat/Km 9 to 24-fold.
► Mutation of Os3BGlu7 Asn245 and Os4BGlu12 His252 to Met decreased these kcat/Km values.