2BER

Y370G Active Site Mutant of the Sialidase from Micromonospora viridifaciens in complex with beta-Neu5Ac (sialic acid).

Summary for 2BER

• Entry DOI: 10.2210/pdb2ber/pdb
• Related: 1EUR 1EUS 1EUT 1EUU 1W8N 1W8O 1WCQ
• Descriptor: BACTERIAL SIALIDASE, N-acetyl-beta-neuraminic acid, SODIUM ION, ... (4 entities in total)
• Functional Keywords: glycosidase, hydrolase, sialidase, beta-propeller, micromonospora viridifaciens
• Biological source: MICROMONOSPORA VIRIDIFACIENS
• Cellular location: Secreted: Q02834
• Total number of polymer chains: 1
• Total formula weight: 64524.76

Authors

Newstead, S.,Watson, J.N.,Bennet, A.J.,Taylor, G.L. (deposition date: 2004-11-30, release date: 2005-04-04, Last modification date: 2024-11-06)

Primary citation

Newstead, S.,Watson, J.N.,Knoll, T.L.,Bennet, A.J.,Taylor, G.L.
Structure and Mechanism of Action of an Inverting Mutant Sialidase.
Biochemistry, 44:9117-, 2005

PubMed 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.

PubMed: 15966735
DOI: 10.1021/BI050517T

Experimental method

X-RAY DIFFRACTION (1.8 Å)