Biochemistry of actinobacterial coproheme decarboxylase (ChdC) from Corynebacterium diphteriae



Until recently, the protoporphyrin-dependent heme biosynthesis pathway was thought to be the major route to synthesize heme in the bacterial world, besides an alternative pathway, which is utilized by Archaea and denitrifying bacteria. In 2015 the coproporphyrin-dependent heme biosynthesis pathway was first described (Dailey et al., 2015) with coproheme decarboxylases (ChdCs) catalyzing the last step in mainly monoderm bacteria (Dailey et al., 2015, Lobo et al., 2015). ChdCs do not have a eukaryotic counterpart. Since many pathogenic monoderm bacteria already display multiple resistances to common antibiotics, ChdCs are a promising target for the development of urgently needed novel antibacterial substances. Moreover, the ability of pathogens to obtain and process (heme) iron is a crucial factor of their virulence and survival (Sheldon & Heinrichs, 2015).

ChdCs catalyse the decarboxylation of two propionate groups of iron coproporphyrin III (coproheme) to form heme b, which is the final product of heme biosynthesis. In this reaction coproheme is substrate and redox co-factor at the same time. The reaction mechanism of this enzyme is not fully understood yet. Most biochemical and biophysical studies on purified ChdCs were performed on enzymes from Firmicutes (Clade 1), e.g. ChdCs from Listeria monocytogenes and Staphylococcus aureus (Hofbauer et al., 2016b, Hofbauer et al., 2016a, Celis et al., 2015, Celis et al., 2017). ChdC from Corynebacterium diphteriae belongs phylogenetically to Clade 2 and exhibits significant differences to Clade 1 ChdCs concerning reaction kinetics and its predicted active site architecture (Pfanzagl et al., 2018). It is known that ChdCs depend on an essential, catalytic tyrosine residue, but the exact mechanism of catalysis and the relevant redox intermediates are unkown. In this project we aim to study the structure-function relationships of ChdC from Corynebacterium diphteriae in order to understand the underlying principles of the radicalic decarboxylation reaction.

Aims and methods.

Homopentameric ChdC from Corynebacterium diphteriae (wild-type and variants) will be heterologously expressed in E. coli. Based on recently solved X-ray structures from other ChdCs and structural models, site-directed mutagenesis of distal and proximal amino acids will be performed. Emphasis will be placed on amino acids involved in coproheme binding and oxidation, redox regulation and internal electron transfer.

Wild-type and mutant proteins will be characterized by a broad set of biochemical/physical methods including (i) X-ray crystallography (in close cooperation with Kristina DJINOVIC-CARUGO from the Department for Structural and Computational Biology, Max F. Perutz Laboratories, University of Vienna), (ii) detailed spectral analysis (UV-Vis, electron paramagnetic resonance and resonance Raman spectroscopy) of proteins, and protein-based radicals in different redox- and spin-states (in cooperation with Sabine VAN DOORSLAER from the Department of Physics, University of Antwerp), (iii) time-resolved multi-mixing UV-Vis stopped-flow studies in order to analyse the kinetics of interconversion and spectroscopic features of relevant redox intermediates, (iv) spectroelectrochemical studies (in cooperation with Gianantonio BATTISTUZZI from the Department of Chemistry, University of Modena and Reggio Emilia, Italy, and Roland LUDWIG from BOKU).

These methods will provide valuable insight into the mechanism of coproheme decarboxylation. Proposed pathways will be analyzed by classical and ab initio molecular dynamics simulations (in cooperation with OOSTENBRINK). Obtained data will provide an excellent basis for potential in silico drug design approaches on these essential players in prokaryotic heme biosynthesis.

Celis, A.I., Gauss, G.H., Streit, B.R., Shisler, K., Moraski, G.C., Rodgers, K.R., Lukat-Rodgers, G.S., Peters, J.W., and DuBois, J.L. (2017) Structure-Based Mechanism for Oxidative Decarboxylation Reactions Mediated by Amino Acids and Heme Propionates in Coproheme Decarboxylase (HemQ). J. Am. Chem. Soc. 139: 1900-1911.
Celis, A.I., Streit, B.R., Moraski, G.C., Kant, R., Lash, T.D., Lukat-Rodgers, G.S., Rodgers, K.R., and DuBois, J.L. (2015) Unusual Peroxide-Dependent, Heme-Transforming Reaction Catalyzed by HemQ. Biochemistry 54: 4022-4032.
Dailey, H.A., Gerdes, S., Dailey, T.A., Burch, J.S., and Phillips, J.D. (2015) Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin. Proc. Natl. Acad. Sci. U S A 112: 2210-2215.
Hofbauer, S., Dalla Sega, M., Scheiblbrandner, S., Jandova, Z., Schaffner, I., Mlynek, G., Djinovic-Carugo, K., Battistuzzi, G., Furtmuller, P.G., Oostenbrink, C., and Obinger, C. (2016a) Chemistry and Molecular Dynamics Simulations of Heme b-HemQ and Coproheme-HemQ. Biochemistry 55: 5398-5412.
Hofbauer, S., Mlynek, G., Milazzo, L., Puhringer, D., Maresch, D., Schaffner, I., Furtmuller, P.G., Smulevich, G., Djinovic-Carugo, K., and Obinger, C. (2016b) Hydrogen peroxide-mediated conversion of coproheme to heme b by HemQ-lessons from the first crystal structure and kinetic studies. FEBSJ. 283: 4386-4401.
Lobo, S.A., Scott, A., Videira, M.A., Winpenny, D., Gardner, M., Palmer, M.J., Schroeder, S., Lawrence, A.D., Parkinson, T., Warren, M.J., and Saraiva, L.M. (2015) Staphylococcus aureus haem biosynthesis: characterisation of the enzymes involved in final steps of the pathway. Mol. Microbiol. 97: 472-487.
Pfanzagl, V., Holcik, L., Maresch, D., Gorgone, G., Michlits, H., Furtmüller, P.G., and Hofbauer, S. (2018) Coproheme decarboxylases - Phylogenetic prediction versus biochemical experiments. Arch. Biochem. Biophys. 640: 27-36.
Sheldon, J.R., and Heinrichs, D.E. (2015) Recent developments in understanding the iron acquisition strategies of gram positive pathogens. FEMS Microbiol. Rev. 39: 592-630.