1WCQ

Mutagenesis of the Nucleophilic Tyrosine in a Bacterial Sialidase to Phenylalanine.

Summary for 1WCQ

• Entry DOI: 10.2210/pdb1wcq/pdb
• Related: 1EUR 1EUS 1EUT 1EUU 1W8N 1W8O
• Descriptor: SIALIDASE, SODIUM ION, GLYCEROL, ... (5 entities in total)
• Functional Keywords: hydrolase, sialidase, micromonospora viridifaciens, hydrolase neuraminidase, glycosidase
• Biological source: MICROMONOSPORA VIRIDIFACIENS
• Cellular location: Secreted: Q02834
• Total number of polymer chains: 3
• Total formula weight: 194435.28

Authors

Newstead, S.,Watson, J.N.,Bennet, A.J.,Taylor, G. (deposition date: 2004-11-19, release date: 2005-10-12, Last modification date: 2024-10-23)

Primary citation

Watson, J.N.,Newstead, S.,Narine, A.A.,Taylor, G.,Bennet, A.J.
Two Nucleophilic Mutants of the Micromonospora Viridifaciens Sialidase Operate with Retention of Configuration by Two Different Mechanisms.
Chembiochem, 6:1439-, 2005

PubMed Abstract: Mutants of the Micromonospora viridifaciens sialidase, Y370E and Y370F, are catalytically active retaining enzymes that operate by different mechanisms. Previous substitutions with smaller amino acids, including Y370D, yielded inverting sialidases. At least one water molecule can fit into the active-site cavity of this mutant and act as a nucleophile from the face opposite the leaving group (Biochemistry 2003, 42, 12 682). Thus, addition of a CH(2) unit (Asp versus Glu) changes the mechanism from inversion back to retention of configuration. Based on Brønsted beta(lg) values, it is proposed that the Y370E mutant reacts by a double-displacement mechanism (beta(lg) on k(cat)/K(m) -0.36+/-0.04) with Glu370 acting as the nucleophile. However, the Y370F mutant (beta(lg) on k(cat)/K(m) -0.79+/-0.12) reacts via a dissociative transition state. The crystal structure of the Y370F mutant complexed with 2-deoxy-2,3-dehydro-N-acetylneuraminic acid shows no significant active-site perturbation relative to the wild-type enzyme.

PubMed: 16206228
DOI: 10.1002/CBIC.200500114

Experimental method

X-RAY DIFFRACTION (2.1 Å)