Nanomolar versus Millimolar Inhibition by Xylobiose-Derived Azasugars:  Significant Differences between Two Structurally Distinct Xylanases

Abstract

The synthesis of xylobiose-derived nitrogen-containing inhibitors of xylanase is described starting with accessible precursors through efficient synthetic schemes. Four disaccharides were identified as powerful competitive inhibitors of the retaining family 10 xylanase, Cex, from Cellulomonas fimi, namely imidazole (K i ) 150 nM), lactam oxime (K i ) 370 nM), isofagomine (K i ) 130 nM), and deoxynojirimycin (K i ) 5800 nM) derivatives of xylobiose. By contrast, none of the compounds inhibited the family 11 xylanase, Bcx, from Bacillus circulans, to an appreciable extent. Two possible explanations are provided for the discrimination exhibited by the imidazole and the lactam oxime. One explanation relates to the different active site locations of the acid/base residue in the two enzymes: anti to the C1-O5 bond for the family 10 Cex and syn for the family 11 Bcx. The other explanation concerns proposed differences in the transition state conformation for the two enzymes: half-chair for Cex and boat for Bcx. The reasons for the difference in inhibition values between Cex and Bcx for the isofagomine and deoxynojirimycin derivatives are less clear-cut but may be ascribed to destabilizing steric and electrostatic interactions between the inhibitors and an essential tyrosine residue in the active site of Bcx.

Biocatalytic preparation of enantioenriched 3,4-dihydroxypiperidines and theoretical study of Candida antarctica lipase B enantioselectivity

“…4 In this sense, optically pure 3,4-dihydroxypiperidine is an interesting structure since the (3R,4R)-trans isomer is an inhibitor of -D-glucoronidase 5 and has been used as intermediate for the preparation of a xylanase inhibitor (Figure 1). 6 Also, this structure is a suitable precursor of the gastroprokinetic agent cisapride. 7 On the other hand, the cis-isomer is an intermediate for the preparation of the antidepressant ifoxetine.…”

Gas phase noncovalent protein complexes that retain solution binding properties: Binding of xylobiose inhibitors to the β-1, 4 exoglucanase from Cellulomonas fimi

“…Cex is a family 10 glycosyl hydrolase, found in the microorganism Cellulomonas fimi. It catalyzes the hydrolytic cleavage of ␤-1,4-linked polymers of xylose [18]. Five high-affinity xylobiose-derived aza-sugars that inhibit the catalytic activity of Cex have been described: imidazole, lactam oxime, deoxynojirimycin, isofagomine lactam, and isofagomine [19].…”

“…Figure 1 shows the structures of the three inhibitors used here, with binding affinity increasing in the order xylobiosyl-deoxynojirimycin (X 2 DNJ), xylobiosyl-isofagomine lactam (X 2 IL), and xylobiosyl-isofagomine (X 2 IF). All inhibitors consist of two sugars: a common distal xylose which binds to the Ϫ2 sub-site of Cex, and a distinct proximal aza-sugar which binds to the Ϫ1 sub-site [18,20]. Cex binds to the proximal and distal sugars through van der Waals interactions, electrostatic interactions, and a series of hydrogen bonds to amino acid side chains.…”

Solution and Gas-Phase H/D Exchange of Protein–Small-Molecule Complexes: Cex and Its Inhibitors

“…Cex (from the micro-organism Cellulomonas fimi) is a family 10 glycosal hydrolase. The catalytic domain of Cex folds as a (α/β) 8 -barrel, and catalyses hydrolytic cleavage of β-1,4 linked polymers of D-xylose [27]. A series of small-molecule inhibitors that noncovalently bind at the active site of the catalytic domain of Cex (Cex-cd) with high affinity have been described [27]: imidazole, lactam oxime, deoxynojirimycin (X 2 DNJ), isofagomine lactam (X 2 IL), and isofagomine (X 2 IF), three of which are used in this work and are shown in Figure 1.…”

“…The catalytic domain of Cex folds as a (α/β) 8 -barrel, and catalyses hydrolytic cleavage of β-1,4 linked polymers of D-xylose [27]. A series of small-molecule inhibitors that noncovalently bind at the active site of the catalytic domain of Cex (Cex-cd) with high affinity have been described [27]: imidazole, lactam oxime, deoxynojirimycin (X 2 DNJ), isofagomine lactam (X 2 IL), and isofagomine (X 2 IF), three of which are used in this work and are shown in Figure 1. These xylobiose-derived nitrogen-containing inhibitors consist of two sugars, a common distal xylose that binds to the −2 sub-site of Cex and a distinct proximal aza-sugar that binds to the −1 sub-site [27,28].…”