Characterisation of Desulfovibrio vulgaris haem b synthase, a radical SAM family member.
An alternative route for haem b biosynthesis is operative in sulfate-reducing bacteria of the Desulfovibrio genus and in methanogenic Archaea. This pathway diverges from the canonical one at the level of uroporphyrinogen III and progresses via a distinct branch, where sirohaem acts as an intermediate precursor being converted into haem b by a set of novel enzymes, named the alternative haem biosynthetic proteins (Ahb). In this work, we report the biochemical characterisation of the Desulfovibrio vulgaris AhbD enzyme that catalyses the last step of the pathway. Mass spectrometry analysis showed that AhbD promotes the cleavage of S-adenosylmethionine (SAM) and converts iron-coproporphyrin III via two oxidative decarboxylations to yield haem b, methionine and the 5'-deoxyadenosyl radical. Electron paramagnetic resonance spectroscopy studies demonstrated that AhbD contains two [4Fe-4S](2+/1+) centres and that binding of the substrates S-adenosylmethionine and iron-coproporphyrin III induces conformational modifications in both centres. Amino acid sequence comparisons indicated that D. vulgaris AhbD belongs to the radical SAM protein superfamily, with a GGE-like motif and two cysteine-rich sequences typical for ligation of SAM molecules and iron-sulfur clusters, respectively. A structural model of D. vulgaris AhbD with putative binding pockets for the iron-sulfur centres and the substrates SAM and iron-coproporphyrin III is discussed.
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Susana A L
SA
Lobo
Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Andrew D
AD
Lawrence
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal.
Célia V
CV
Romão
Centre for Molecular Processing, School of Biosciences, University of Kent, Canterbury, Kent CT2 7NJ, UK.
Martin J
MJ
Warren
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: miguel@itqb.unl.pt.
Miguel
M
Teixeira
Instituto de Tecnologia Química e Biológica António Xavier, Universidade Nova de Lisboa, Av. da República, 2780-157 Oeiras, Portugal. Electronic address: lst@itqb.unl.pt.
Lígia M
LM
Saraiva
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000378:metabolism*; D002384:Catalysis; D016969:Desulfovibrio vulgaris / Q000201:enzymology* / Q000254:growth & development; D004578:Electron Spin Resonance Spectroscopy; D006418:Heme / Q000096:biosynthesis*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D011487:Protein Conformation; D012436:S-Adenosylmethionine / Q000378:metabolism; D017386:Sequence Homology, Amino Acid; D013056:Spectrophotometry, Ultraviolet
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C000589762:haem b synthase, Desulfovibrio vulgaris; D006418:Heme; D012436:S-Adenosylmethionine
Alternative haem biosynthesis; Desulfovibrio; Iron-coproporphyrin III; S‐adenosylmethionine; iron-sulfur protein