Fig. 3.

(A) Surface representation of the MHV-DVIM HE (Left) and RCoV-NJ HE (Right) catalytic sites in complex with 9-O-Ac-Sia [docked with Autodock4 (55)] and 4-N-Ac-Sia (crystal complex), respectively. (B) Surface representation of the catalytic sites of MHV-DVIM HE (Left) and RCoV-NJ HE (Right). The active-site Ser⁴⁴ in MHV-DVIM HE already adopts the “active” rotamer observed in HEF (18, 39, 40); For RCoV-NJ HE crystallized as an inactive Ser-to-Ala mutant, a Ser side chain with active rotamer was introduced using COOT. The P1 and P2 pockets are highlighted by dashed circles; approximate distances between pockets, as measured from the centers, are indicated. (C) Binding topology of αNeu4,5,9Ac₃ in type I (Left) and type II (Right) esterases. The P1 and P2 pockets accommodating the O- and N-acetyl moieties are shown schematically. αNeu4,5,9Ac₃ is shown in stick representation and colored as in Fig. 2C. Asterisks indicate the position of the O₂ atom through which Sias are glycosidically linked. The distances between 5-N- and 9-O- or 4-O-Ac methyl groups are shown. (D) RCoV-NJ HE Arg³⁰⁷ is not essential for sialate-4-O-acetylesterase activity. Ser⁴⁰Ala is a catalytically inactive mutant. Receptor destruction was assessed as in Fig. 1B. For a comparison with type I HEs, see Fig. 2E. (E) Arg³⁰⁷Ala substitution in RCoV-NJ HE does not affect activity toward the synthetic substrate pNPA. Enzymatic activity shown as percentage of wild-type activity. (F) Hydrogen bonding of the sialate-5-N-acyl carbonyl oxygen and amide nitrogen with RCoV-NJ HE Ser⁷⁴ and His³³⁶, respectively, as observed in the crystal complex, indicated as in Fig. 2C. Hydrophobic contacts between Tyr⁴⁶ and the Sia-5-N-acyl methyl group are shown as thin gray lines.