Mutagenesis of the Conserved Active-Site Tyrosine Changes a Retaining Sialidase into an Inverting Sialidase

Abstract

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase changes the mechanism of catalysis from retention of anomeric configuration to an unprecedented inverting mechanism in which water efficiently functions as the nucleophile. Three mutants, Y370A, Y370D, and Y370G, were produced recombinantly in Escherichia coli, and all are catalytically active against the activated substrate 4-methylumbelliferyl alpha-D-N-acetylneuraminide. The Y370D mutant was also shown to catalyze the hydrolysis of natural substrate analogues such as 3'-sialyllactose. A comparison of the pH-rate profiles for the wild-type and the Y370D mutant sialidase reveals no major differences, although with respect to the kinetic term k(cat)/K(m), an ionized form of the aspartate-370 enzyme is catalytically compromised. For the wild-type enzyme, the value of the Brønsted parameter beta(lg) on k(cat) is 0.02 +/- 0.03, while for the Y370D mutant sialidase beta(lg) = -0.55 +/- 0.03 for the substrates with bad leaving groups. Thus, for the wild-type enzyme, a nonchemical step(s) is rate-limiting, but for the tyrosine mutant cleavage of the glycosidic C-O bond is rate-determining. The Brønsted slopes derived for the kinetic parameter k(cat)/K(m) display a similar trend (beta(lg) -0.30 +/- 0.04 and -0.74 +/- 0.04 for the wild-type and Y370D, respectively). These results reveal that the tyrosine residue lowers the activation free energy for cleavage of 6'-sialyllactose, a natural substrate analogue, by more than 24.9 kJ mol(-1). Evidence is presented that the mutant sialidases operate by a dissociative mechanism, and the wild-type enzyme operates by a concerted mechanism.

Structural and Kinetic Analysis of Two Covalent Sialosyl-Enzyme Intermediates on Trypanosoma rangeli Sialidase

“…However, as the authors stress (31), the high residual activities for the nucleophile mutants might be a trait of this particular bacterial sialidase, because equivalent mutations in other sialidases result in complete inactivation. Furthermore, the catalytic rates of the mutants are critically dependent on the aglyconeleaving group ability in contrast to the behavior of the wild-type enzyme, which shows higher k cat values (Ͼ10 4 ) when working with natural substrates, such as ␣-2,6-and ␣-2,3-sialyl-lactose (29,31). In the absence of the naturally selected nucleophilic tyrosine, which is strictly conserved in all known exosialidases, compensation by alternative chemical groups appears to take place on the M. viridifaciens mutants, with reaction occurring via several different mechanisms (29,31).…”

Influenza neuraminidase operates via a nucleophilic mechanism and can be targeted by covalent inhibitors

“…Retaining NA mechanisms, on the other hand, appear to be quite distinct. Biochemical analysis of NA from various microorganisms has revealed that a conserved active-site tyrosine can function as a nucleophile and may be the most important catalytic residue [17][18][19] .…”

Extremophilic Carbohydrate Active Enzymes (CAZymes)

“…A tyrosine residue is a neutral nucleophile, but requires a general base to enhance its nucleophilicity. This mechanism was implied from X-ray structures, and was supported by experiments involving trapping of the intermediate with fluoro sugars followed by peptide mapping and then crystallography [6,7], and also through mechanism studies on mutants [8] (Figure 7). …”

“…As members of Clan GH-A they have a classical (α/β) 8 TIM barrel fold with the two key active site glutamic acids being approximately 200 residues apart in sequence and located at the C-terminal ends of β-strands 4 (acid/base) and 7 (nucleophile) [15,16].…”