Premium
KINETIC STUDIES OF NEISSERIA MENINGITIDIS SEROGROUP W CAPSULE POLYMERASE USING BIOLUMINESCENCE‐BASED ASSAYS
Author(s) -
Moghaddam Laleh Sheikhi,
McCarthy Pumtiwitt
Publication year - 2020
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.2020.34.s1.01904
Subject(s) - neisseria meningitidis , sialic acid , enzyme , galactose , acceptor , chemistry , biochemistry , polymerase , stereochemistry , microbiology and biotechnology , biology , bacteria , physics , genetics , condensed matter physics
Neisseria meningitidis is a Gram‐negative bacterium that causes meningitis. Our overall goal is increased understanding of the N. meningitidis serogroup W capsule polymerase. This enzyme creates the capsular polysaccharide found in this serogroup. During the reaction, the enzyme transfers galactose and sialic acid from two nucleotide donor sugars (UDP‐Galactose and CMP‐Sialic Acid) to an acceptor. This capsule polymerase can be used as a new tool for glycoconjugate vaccine development. In this work, we describe our efforts to determine kinetic parameters of the enzyme which are currently unknown. We have optimized commercially available bioluminescence‐based assays to investigate activity of the enzyme (CMP‐Glo and UDP‐Glo by Promega). Reactions were performed to determine K m and V max values in a 10‐minute reaction. K m and V max values of 629.2 μg/mL and 0.8965 μM/min were determined for the acceptor (hydrolyzed serogroup W polysaccharide). To simplify our studies, we have recently moved to use of a homogeneous acceptor, a sialic acid trimer (DP3) and focused strictly on the UDP‐Glo assay. In our studies with this acceptor, we confirm an increase in product formation with increasing enzyme amounts (0–1250 ng enzyme). K m and V max values were determined for UDP‐Galactose (44.61 μM and 0.009457 μM/min) and DP3 acceptor (2984.2 μM and 0.01099 μM/min). In future work, we will perform kinetic studies with a modified acceptor (DP3‐Galactose) to determine K m and V max for this acceptor and CMP Sialic acid. Support or Funding Information NIH 5SC2GM125517‐02NIH UL1GM118973