Structure and Mechanism of Action of an Inverting Mutant Sialidase

Abstract

Mutagenesis of the conserved tyrosine (Y370) of the Micromonospora viridifaciens sialidase to small amino acids changes the mechanism of catalysis from retention of anomeric configuration to inversion [Watson, J. N., et al. (2003) Biochemistry 42, 12682-12690]. For the Y370G mutant enzyme-catalyzed hydrolysis of a series of aryl sialosides and 3'-sialyllactose, the derived Brønsted parameters (beta(lg)) on k(cat) and k(cat)/K(m) are -0.63 +/- 0.05 and -0.80 +/- 0.08, respectively. Thus, for the Y370G enzyme, glycosidic C-O bond cleavage is rate-determining. Analysis of the activity of the Y370G mutant and wild-type enzymes against a substrate [3,4-dihydro-2H-pyrano[3,2-c]pyridinium alpha-d-N-acetylneuraminide (DHP-alphaNeu5Ac)] whose hydrolysis cannot be accelerated by acid catalysis is consistent with these reactions proceeding via S(N)1 and S(N)2 mechanisms, respectively. The overall structure of the Y370G mutant sialidase active site is very similar to the previously reported wild-type structure [Gaskell, A., et al. (1995) Structure 3, 1197-1205], although removal of the tyrosine residue creates two significant changes to the active site. First, the anomeric oxygen atom of the hydrolysis product (beta-N-acetylneuraminic acid) and four water molecules bind in the large cavity created by the Y370G mutation. Second, the side chain of Asn310 moves to make a strong hydrogen bond to one of the bound water molecules.

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

“…Recent work on the sialidase from Micromonospora viridifaciens (29,30) shows that mutation of the catalytic tyrosine frequently results in detectable residual activity. Although in terms of catalytic efficiency (k cat /K m ) the best mutant (Y3 D) is 70 times less proficient than the wild type (ϳ10 4 times for the worst mutant Y3 H, naturally present in trypanosomal members of the family), it is indeed astonishing to find high values of k cat ranging from 400 to only 3.5 times lower rates than that of the non-mutated enzyme.…”

Structural Basis for the Recognition and Cleavage of Polysialic Acid by the Bacteriophage K1F Tailspike Protein EndoNF

“…A similar effect has been observed in exosialidases; substitution of the catalytic tyrosine enabled a water molecule to act as the nucleophile in the cleavage reaction. 33,34 In summary, His350 is crucial to the reaction mechanism and may serve as a general acid in the initial attack of the glycosidic bond. Structural and biochemical data provide convincing evidence to explain polySia cleavage by endoNF through an S N 1-type reaction mechanism, resulting in an inversion of the stereochemistry at the anomeric center.…”

Galactose recognition by the carbohydrate-binding module of a bacterial sialidase

“…Data were processed and scaled with MOSFLM and SCALA from the CCP4 (v.5.0.1) suite of programs (Collaborative Computational Project, Number 4, 1994). Several crystal forms of the M. viridifaciens sialidase have been obtained previously: a monoclinic form for a Y370G mutant (Newstead et al, 2005), an orthorhombic form for a D92G mutant (Watson et al, 2004) and a trigonal form for a Y370F mutant (Watson et al, 2005). For the galactose soak, crystals of the active-site mutant (E260A) were grown in conditions similar to the Y370G orthorhombic crystals, yet crystallized in space group P3 2 21, with unit-cell parameters a = b = 141.46, c = 158.89 Å .…”