Heme is an essential cofactor in most organisms, this includes bacteria and mammals. The coproporphyrin-dependent heme biosynthesis pathway is specific to Gram positive bacteria and was only discovered in 2015. Before this discovery Gram positive bacteria were assumed to synthesise heme using the same heme synthesis pathway as mammals. As a result, the enzymes within this pathway have not been fully characterised and provide novel targets for antibiotics.
Experiments were completed to investigate the protein-protein interactions between HemH and HemQ, two consecutive enzymes in the pathway as they are covalently linked in P. acnes. This used SEC and showed that truncations for the P. acnes HemH-Q protein (HemHL and HemQS) interacted 1:1 whereas other non-covalently linked HemH and HemQ proteins didn’t have observable protein-protein interactions.
Kinetic investigation of the wildtype B. subtilis and S. aureus HemH proteins with their endogenous substrate (coproporphyrin) was completed as previous kinetic analysis of B. subtilis HemH used analogues of protoporphyrin IX in the kinetic assays. A combination of spectroscopic techniques were utilised and they show that the two proteins are very active and behave in a broadly similar way to each other. Stopped flow fluorescence spectroscopy proved a useful tool for in depth kinetic investigation into the enzymatic mechanism of coproporphyrin ferrochelatase. Rates constants for enzyme/porphyrin isomerisation, metal chelation and binding constants for substrate binding were estimated using this technique.
Finally, functionally important B. subtilis HemH active site mutants (K87A, H88A, E264A/Q) were characterised using similar techniques described for wildtype characterisation. K87A/H88A and E264A/Q are on the non-conserved and conserved active site face, respectively. This shows that whilst K87A, E264A/Q are relatively inactive they are capable of binding coproporphyrin and activity is diminished after binding. H88A has activity comparable to wildtype with weakened coproporphyrin binding and better at iron binding and chelation. This research could provide details for rational drug design of antibiotics that specifically target Gram positive bacteria.
