Investigation of bioluminescence-based assays for determination of kinetic parameters for the bifunctional Neisseria meningitidis serogroup W capsule polymerase

Abstract

Objective Neisseria meningitidis is a Gram-negative bacterium that causes meningitis. N. meningitidis serogroup W (NmW) capsule polymerase synthesizes capsular polysaccharide of this serogroup. This enzyme could be a tool for meningococcal glycoconjugate vaccine development. Our long-term goal is to control activity of the NmW capsule polymerase for production of defined carbohydrates for vaccines. The enzyme lacks a simple, high-throughput activity assay. Here, we describe the use of high-throughput bioluminescence assays (CMP-Glo and UDP-Glo by Promega) to investigate NmW capsule polymerase activity. These assays detect free nucleotides produced during transfer of sugar from UDP-Galactose and CMP-Sialic Acid to an acceptor. Kinetic studies using NmW hydrolyzed polysaccharide (PS) acceptor are described as well as preliminary work with a sialic acid trimer (DP3) acceptor. Results In CMP-Glo kinetic studies, with constant donor (80 µM) and varied NmW hydrolyzed polysaccharide (0–2000 µg/mL), a Km of 629.2 ± 101.4 µg/mL and a Vmax of 0.8965 ± 0.05823 µM/min was obtained. Using UDP-Glo, Km and Vmax values of 13.84 ± 9.675 µM and 0.6205 ± 0.1331 µM/min were obtained with varied CMP-NeuNAc (0–80 µM) and constant acceptor (400 µg/mL) and UDP-Gal (80 µM). This is the first report of using bioluminescence assays for NmW kinetics.

The Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118)

“…These results demonstrate that the purified fusion enzyme is active in vitro and also shows some substrate promiscuity with respect to glycosyl acceptors, with both PNP-Glcp and Galp acceptors used by the enzyme with comparable efficiency. We subsequently used a CMP-Glo™ assay [20] to measure kinetic…”

“…These results demonstrate that the purified fusion enzyme is active in vitro and also shows some substrate promiscuity with respect to glycosyl acceptors, with both PNP‐Glc p and Gal p acceptors used by the enzyme with comparable efficiency. We subsequently used a CMP‐Glo™ assay [20] to measure kinetic parameters of Im9‐KpsS1 (Supplementary Figures S9 and S11) and determined the enzyme bound CMP‐Pse5Ac7Ac 3 with a K m of approximately 105 μM, which is comparable to the donor affinity of other bacterial sialyltransferases [21] …”

Biological functions of sialic acid as a component of bacterial endotoxin

“…Studies on the structure and function of bacterial antigens, that mimic tissue epitopes, may have a great impact on effective and safe vaccines manufacture ( Sheikhi Moghaddam et al, 2021 ). It can also explain the mechanisms of tolerance to pathogenic factors, overcoming this tolerance and inducing production of autoantibodies.…”

The Retaining Pse5Ac7Ac Pseudaminyltransferase KpsS1 Defines a Previously Unreported glycosyltransferase family (GT118)

“…These results demonstrate that the purified fusion enzyme is active in vitro and also shows some substrate promiscuity with respect to glycosyl acceptors, with both PNP-Glcp and Galp acceptors used by the enzyme with comparable efficiency. We subsequently used a CMP-Glo™ assay [20] to measure kinetic parameters of Im9-KpsS1 (Supplementary Figures S9 and S11) and determined the enzyme bound CMP-Pse5Ac7Ac 3 with a K m of approximately 105 μM, which is comparable to the donor affinity of other bacterial sialyltransferases. [21] Although these enzyme activity assays provided evidence of turnover, the limited availability of pure CMP-Pse5Ac7Ac 3 donor restricted the scale on which these reactions could be performed and precluded NMR characterisation of the products.…”