Two Nucleophilic Mutants of the Micromonospora viridifaciens Sialidase Operate with Retention of Configuration by Two Different Mechanisms

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.

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).…”

“…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). Although artificial, these results are fully consistent with the evolutionary concept of adaptation, whereby mutations on a particular residue become acceptable as soon as alternative reactive groups relieve the selective pressure.…”

“…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.…”

Study of the Enzymatic Activity of Arthrobacter ureafaciens Neuraminidase by Isothermal Titration Calorimetry

“…Bacterial sialidase from Micromonospora viridifaciens ( M.v. ) contains a galactose binding site . Homologous sequence analysis of the M.v.…”