Determination of the Kinetic and Chemical Mechanism of a Unique Peptidoglycan Recycling Enzyme with Dual Hydrolase and Kinase Functionality: Anhydromuramic Acid Kinase, AnmK

Abstract

<p>Antibiotic resistance is a crisis in modern society causing increasing rates of bacterial infections impervious to current therapies. To this end, new targets for antimicrobial treatment must be pursued. Peptidoglycan recycling is an understudied key life process in the bacterial cell where over 60% of cell wall materials are reused during each turnover. Anhydro-­N-acetylmuramic acid kinase, AnmK is a novel enzyme in this pathway catalyzing the conversion of anhydro-­N-acetylmuramic acid to N-acetylacetylmuramic acid-­6-­phosphate in the presence of magnesium and ATP. Previously, several crystal structures of AnmK have been solved providing insights into the catalytic mechanism, but until this point, no extensive work has been done. The goal of this work is to determine the chemical and kinetic mechanisms of AnmK. This will be completed using a continuous assay for dual hydrolysis and phosphorylation activity as well as a novel assay for carbohydrate hydrolysis. Substrate interactions will be probed using previous crystal structures as a guide. Finally, pre-steady-­state studies will conclude the mechanistic studies giving a full depiction of AnmK catalysis. </p><p>The kinetic and chemical mechanisms of AnmK are studied in the steady-­state using a continuous assay format where bisubstrate kinetics as well as inhibitor studies were performed as well as substrate specificity studies. pH rate profiles and solvent isotope exchange were used to determine the residues involved in catalysis. </p><p>The concerted or stepwise nature of hydrolysis and phosphorylation is a main question of this work. A novel assay using glucose oxidase was executed to trap any chemical intermediates formed in a potential stepwise reaction. This assay was used on wild-type AnmK as well as a variety of mutants. Through these experiments, two key residues in phosphoryl transfer are identified and used to partially decouple hydrolysis and phosphorylation. </p><p>Mechanistic studies were continued by investigating the pre-­steady-­state kinetics of AnmK using a quench flow apparatus. Wild-type AnmK showed no appearance of a chemical intermediate during timepoints as short as 10 ms and also showed a linear formation of product with a catalytic rate analogous to the steady-­state rate. These results indicate that AnmK undergoes a concerted, one-step catalytic mechanism with no chemical intermediate. AnmK E330A formed both hydrolysis product, as well as hydrolysis and phosphorylation product in the pre-steady-­state agreeing with the previous results that hydrolysis and phosphorylation had been partially decoupled. </p><p>These results show the chemical and kinetic mechanism of a novel enzyme with previously undocumented concomitant hydrolysis and phosphorylation. This work provides further understanding of carbohydrate-modifying enzymes as well as the peptidoglycan recycling pathway. A better understanding of peptidoglycan biosynthesis and recycling could lead to novel antimicrobial therapies in the future.</p>