Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in α-proteobacteria.pdf (9.81 MB)
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Identification and characterization of the 'missing' terminal enzyme for siroheme biosynthesis in α-proteobacteria.

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journal contribution
posted on 24.10.2019, 10:14 by S Bali, S Rollauer, P Roversi, E Raux-Deery, SM Lea, MJ Warren, SJ Ferguson
It has recently been shown that the biosynthetic route for both the d1 -haem cofactor of dissimilatory cd1 nitrite reductases and haem, via the novel alternative-haem-synthesis pathway, involves siroheme as an intermediate, which was previously thought to occur only as a cofactor in assimilatory sulphite/nitrite reductases. In many denitrifiers (which require d1 -haem), the pathway to make siroheme remained to be identified. Here we identify and characterize a sirohydrochlorin-ferrochelatase from Paracoccus pantotrophus that catalyses the last step of siroheme synthesis. It is encoded by a gene annotated as cbiX that was previously assumed to be encoding a cobaltochelatase, acting on sirohydrochlorin. Expressing this chelatase from a plasmid restored the wild-type phenotype of an Escherichia coli mutant-strain lacking sirohydrochlorin-ferrochelatase activity, showing that this chelatase can act in the in vivo siroheme synthesis. A ΔcbiX mutant in P. denitrificans was unable to respire anaerobically on nitrate, proving the role of siroheme as a precursor to another cofactor. We report the 1.9 Å crystal structure of this ferrochelatase. In vivo analysis of single amino acid variants of this chelatase suggests that two histidines, His127 and His187, are essential for siroheme synthesis. This CbiX can generally be identified in α-proteobacteria as the terminal enzyme of siroheme biosynthesis.

Funding

This work was funded by research Grant BB/I021736/1 from the Biotechnology and Biological Sciences Research Council to S.J.F and M.J.W. S.E.R is supported by a Wellcome Trust Studentship (Ref:086833). Work in S.M.L.'s group is funded by the James Martin 21st Century School Vaccine Design Institute and by a Wellcome Trust Senior Investigator award (Ref:100298). S.B. would like to thank Dr Matthew Sullivan from University of East Anglia for the kind gift of pK18mobsacB plasmid and the gene deletion protocol. The authors have no conflict of interest to declare.

History

Citation

Molecular Microbiology, 2014, 92 (1), pp. 153-163

Author affiliation

/Organisation/COLLEGE OF LIFE SCIENCES/Biological Sciences/Molecular & Cell Biology

Version

VoR (Version of Record)

Published in

Molecular Microbiology

Publisher

Wiley

eissn

1365-2958

Acceptance date

04/02/2014

Copyright date

2014

Available date

24/10/2019

Publisher version

https://onlinelibrary.wiley.com/doi/full/10.1111/mmi.12542

Notes

Additional supporting information may be found in the online version of this article at the publisher’s web-site.

Language

en