This research investigated N-acetylmannosamine-6-phosphate 2-epimerase (NanE) from methicillin-resistant Staphylococcus aureus (MRSA). NanE catalyses the epimerisation of ManNAc-6P into GlcNAc-6P within the sialic acid degradation pathway. The mechanism by which NanE performs catalysis is controversial. A previous study suggested that NanE uses a deprotonation/reprotonation mechanism that is common for epimerase enzymes1. However, evidence to support this idea is lacking. This research combined structural and kinetic analysis to provide support for a novel alternative, whereby the substrate mediates catalysis in a proton displacement mechanism.
A mutant NanE (E180A) was designed to test the catalytic mechanism and was recombinantly expressed and purified. Biophysical analysis suggested that it has subtly different properties to wildtype NanE in terms of the ratio of secondary structure and the thermal melting properties. Kinetic analysis demonstrated that the mutant has a ~2800-fold reduction in enzyme efficiency relative to the wildtype. The structure of the mutant enzyme was solved with bound substrate, which suggests that E180 may also have a role in orienting the substrate for catalysis.
This research is of interest to the medical sector as understanding the catalytic mechanism of MRSA NanE has important implications for the design of drugs to target the enzyme and treat MRSA infections.