Biochemistry of pathogenic bacteria: Isobacteriochlorin heme d1 biosynthesis in Pseudomonas aeruginosa
Final Report Abstract
The tetrapyrrole cofactor heme d1 plays an essential role for anaerobic energy generation in the opportunistic human pathogen Pseudomonas aeruginosa. The enzymes involved in heme d1 biosynthesis are encoded by the nir genes located in the nir-operon in P. aeruginosa. Within this project (“Biochemistry of pathogenic bacteria: Isobacteriochlorin heme d1 biosynthesis in Pseudomonas aeruginosa”) we elucidated the previously unknown functions of several Nir proteins involved in heme d1 biosynthesis. Recombinant NirE from P. aeruginosa was produced in E. coli, purified and biochemically characterized as the S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase required for heme d1 biosynthesis in P. aeruginosa. Further, NirE was crystallized in the presence of S- adenosyl-L-homocysteine and uroporphyrinogen III and the crystal structure of the enzymesubstrate complex was solved to 2.0 Å resolution. The function of the amino acid residues located within the active site of NirE was studied through site directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants. Recombinant NirD, NirL, NirG and NirH from P. aeruginosa were produced in E. coli and purified. Through co-production and co-purification we showed that hetero-protein complexes are formed between NirD and NirL (NirD/L) and NirG and NirH (NirG/H). The NirD/L/G/H proteins were characterized as DNA-binding proteins which are involved in the transcriptional regulation of the nirJEN genes. Further, using a newly established enzyme activity assay we showed that NirD/L/G/H also acts as siroheme decarboxylase during heme d1 biosynthesis. We found that NirD/L catalyzes the first decarboxylation reaction and NirG/H the second. The siroheme decarboxylase NirDL from Hydrogenobacter thermophilus was crystallized and its structure solved. The overall structure of NirDL strikingly resembles the structures of transcriptional regulators of the Lrp/AsnC-family hinting toward a novel and unique bifunctional enzyme / transcriptional regulator. Moreover, the structure of NirDL in complex with iron-uroporphyrin III provided novel insights into the active site architecture of the enzyme. In a newly developed project we investigated the alternative heme biosynthesis pathway in archaea which shares several common steps with the heme d1 biosynthesis route. Using a bioinformatics approach we identified potential heme biosynthesis enzymes in archaea and we biochemically characterized the SUMT and the precorrin-2 dehydrogenase from Methanosarcina barkeri. Subsequently, we functionally characterized the enzymes AhbAB, AhbC and AhbD responsible for the conversion of siroheme to heme via the intermediates didecarboxy-siroheme and ironcoproporphyrin III. The NirD/L/G/H and AhbD proteins are currently further characterized with the support from the DFG.
Publications
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(2009) The Pseudomonas aeruginosa nirE gene encodes the S- adenosyl-L-methionine dependent uroporphyrinogen III methyltransferase required for heme d1 biosynthesis. FEBS J., 276, 5973-5982
Storbeck, S., Walther, J., Müller, J., Parmar, V., Schiebel, H.M., Kemken, D., Dülcks, T., Warren, M.J. and Layer, G.
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(2010) A novel pathway for the biosynthesis of heme in Archaea: Genome-based bioinformatic predictions and experimental evidence. Archaea, 2010, 175050
Storbeck, S., Rolfes, S., Raux-Deery, E., Warren, M.J., Jahn, D. and Layer, G.
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(2011) Biosynthesis of siroheme, cofactor F430 and heme d1. Handbook of Porphyrin Science, 19, 111-138
Layer, G. and Warren, M.J.
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(2011) Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine dependent uroporphyrinogen III methyltransferases. J. Biol. Chem., 286, 26754-26767
Storbeck, S., Saha, S., Krauße, J., Klink, B., Heinz, D.W. and Layer, G.
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(2011) Tetrapyrrolbiosynthese in denitrifizierenden Bakterien und Archaea. BIOspektrum, 06/11, 637-644
Storbeck, S. and Layer, G.
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The alternative route to heme in the methanogenic archaeon Methanosarcina barkeri. Archaea
Volume 2014, Article ID 327637, 13 pages
Melanie Kühner, Kristin Haufschildt, Alexander Neumann, Sonja Storbeck, Judith Streif, and Gunhild Layer
