Noncanonical coproporphyrin-dependent bacterial heme biosynthesis pathway that does not use protoporphyrin

Abstract

It has been generally accepted that biosynthesis of protoheme (heme) uses a common set of core metabolic intermediates that includes protoporphyrin. Herein, we show that the Actinobacteria and Firmicutes (high-GC and low-GC Gram-positive bacteria) are unable to synthesize protoporphyrin. Instead, they oxidize coproporphyrinogen to coproporphyrin, insert ferrous iron to make Fe-coproporphyrin (coproheme), and then decarboxylate coproheme to generate protoheme. This pathway is specified by three genes named hemY, hemH, and hemQ. The analysis of 982 representative prokaryotic genomes is consistent with this pathway being the most ancient heme synthesis pathway in the Eubacteria. Our results identifying a previously unknown branch of tetrapyrrole synthesis support a significant shift from current models for the evolution of bacterial heme and chlorophyll synthesis. Because some organisms that possess this coproporphyrin-dependent branch are major causes of human disease, HemQ is a novel pharmacological target of significant therapeutic relevance, particularly given high rates of antimicrobial resistance among these pathogens.

Keywords: Gram-positive bacteria; HemN; HemQ; coproporphyrin; heme synthesis.

Conflict of interest statement

The authors declare no conflict of interest.

Figures

Fig. 1.

Currently characterized tetrapyrrole biosynthesis pathways. The core enzymes present in all tetrapyrrole-synthesizing organisms are shown in the blue box. The protoporphyrin-independent, ancestral heme biosynthesis pathway of the Firmicutes and Actinobacteria described in the current study is shown in orange. The primitive heme pathway of the archaea and sulfate-reducing bacteria is shown in the violet box, and the modern heme pathway of Proteobacteria and eukaryotes is shown in the red box. Enzyme abbreviations are those abbreviations currently in acceptance and are shown with single solid lines. Pathways involving more than a single enzyme are shown as double-line arrows. The dashed line represents a proposed pathway for chlorophyll synthesis in bacteria that do not use protoporphyrin. ALA, 5-aminolevulinic acid; COPRO, coproporphyrin; COPRO’GEN, coproporphyrinogen; GltR, glutamyl-tRNA reductase; Glut-tRNA, glutamyl-tRNA; GSAMS, Glu-1-semialdehyde-2,1-aminomutases; HMB, hydroxymethylbilane; PBG, porphobilinogen; PROTO, protoporphyrin; PROTO’GEN, protoporphyrinogen; succCoA, succinyl-coenzymeA; URO’GEN, uroporphyrinogen.

Fig. 2.

HPLC chromatograms of reaction products of HemY/HC/Q reactions. Assays were run as described in the main text. Elution time is shown on the x axis, and absorbance is shown on the y axis. (A) Reaction products from P. acnes HemY + HemH + HemQ with coproporphyrinogen and iron as substrates are shown in the first line (cyan). Reaction products from assay with coproporphyrin and iron as substrates with 50 μM FMN present are shown in the second line (green). Protohemin (blue) and coprohemin (black) standards are shown in the third and fourth lines, respectively. (B) Assay products from M. tuberculosis HemQ assayed with coprohemin as a substrate in the presence of 50 μM H2O2 (blue) or 50 μM FMN (black). (C) Assay products from M. tuberculosis HemQ that was not heme-loaded (blue) or was loaded with heme and then assayed with coprohemin as a substrate in the presence of 50 μM FMN. The mass spectrum results yielded 752.2 (coproheme + formate) for peak 1, 706.2 (monovinyl, monopropionyl heme intermediate + formate) for peak 2, and 660.2 (protoheme + formate) for peak 3.

Fig. 3.

Reaction catalyzed by the enzymes HemY, HemH, and HemQ (details are provided in main text). Coproporphyrinogen III (A), coproporphyrin III (B), coproheme III (C), and protoheme IX (D) are shown. Pyrrole ring lettering and side-chain numbering are shown on protoheme IX.

Fig. 4.

Co-occurrence of HemF, HemN, HemQ, AhbD, and HemY. A Venn diagram illustrates the overlap of the four decarboxylases and HemY. The values shown in the table represent the percentage of each of the four decarboxylases as it is found to occur within the same genome with any of the other carboxylases. Also listed are the occurrence of each decarboxylase with HemY and the total number of genomes in the selection containing each decarboxylase. It should be noted that all heme-synthesizing Gram-positive bacteria possess HemY but few Gram-negative (Proteobacteria) bacteria have a hemY gene.