Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2020 Feb 13;11(1):864.
doi: 10.1038/s41467-020-14722-1.

Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a common active site

Affiliations

Siroheme synthase orients substrates for dehydrogenase and chelatase activities in a common active site

Joseph M Pennington et al. Nat Commun. .

Abstract

Siroheme is the central cofactor in a conserved class of sulfite and nitrite reductases that catalyze the six-electron reduction of sulfite to sulfide and nitrite to ammonia. In Salmonella enterica serovar Typhimurium, siroheme is produced by a trifunctional enzyme, siroheme synthase (CysG). A bifunctional active site that is distinct from its methyltransferase activity catalyzes the final two steps, NAD+-dependent dehydrogenation and iron chelation. How this active site performs such different chemistries is unknown. Here, we report the structures of CysG bound to precorrin-2, the initial substrate; sirohydrochlorin, the dehydrogenation product/chelation substrate; and a cobalt-sirohydrochlorin product. We identified binding poses for all three tetrapyrroles and tested the roles of specific amino acids in both activities to give insights into how a bifunctional active site catalyzes two different chemistries and acts as an iron-specific chelatase in the final step of siroheme synthesis.

PubMed Disclaimer

Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1
Fig. 1. Overview of siroheme synthesis by the CysG homodimer.
a Chemical structures and scheme for the transformation from precorrin-2 to siroheme. b Domain architecture of CysG. The Rossmann fold (R) is colored green throughout; the dimerization domain (d) is colored pink throughout; the all-helical domain (h) is purple throughout; and CysGA is blue throughout. Chain 1 of the dimer is shaded dark throughout and chain 2 of the dimer is shaded light throughout.
Fig. 2
Fig. 2. CysGB architecture and tetrapyrrole binding.
a CysGB is a homodimer with asymmetric cavities between the Rossmann fold (R) of one subunit and the helical domain (h) of the other, joined by a dimerization domain (d). The N- and C-termini are labeled for each and the SUMT is not shown for simplicity. The domains and subunits are colored as in 1b. b The Rossmann fold and helical domains are orthogonal to one another. c The helical domain has a different conformation in the two subunits, resulting in one cavity that is closed and another that is open. d Precorrin-2 binds in the closed active site with strong density for the carboxylates but weaker density for the core ring. The tetrapyrrole binds with an average B-factor of 53.0 Å2 (compared with an overall B-factor for the structure of 50.9 Å2 and for local side chains of 51.9 Å2) and occupancy at 0.5. e Sirohydrochlorin binds to the closed active site with strong density. The tetrapyrrole binds with an average B-factor of 40.0 Å2 (compared with an overall B-factor for the structure of 51.3 Å2 and for local side chains of 48.6 Å2) and occupancy of 1.0. f Co-sirohydrochlorin binds to the open active site, nearly orthogonal to its conformation in the closed active site. The tetrapyrrole binds with an average B-factor of 77.3 Å2 (compared with an overall B-factor for the structure of 77.2 Å2 and for local side chains of 78.3 Å2) and occupancy at 0.5. All Polder omit fofc maps are shown at 2σ (gray) and 3σ (blue).
Fig. 3
Fig. 3. Charged pockets bind the tetrapyrrole.
a Extensive contacts coordinate sirohydrochlorin. b Charge–charge side chain and hydrogen bond main chain interactions coordinate each corner of the tetrapyrrole. c, d The active site is predominantly positively charged (blue) to counter the highly negatively charged sirohydrochlorin, except for the negative charges from D104 and D262 (red) and hydrophobic pockets that position C2 and C7. The electrostatic surface was calculated with UCSF Chimera’s Electrostatic Surface Color tool.
Fig. 4
Fig. 4. CysG variants respond differently to cobalt challenge.
a ANOVA pairwise comparison in a two-tailed test of CysG-deficient E. coli expressing each variant (n = 18, CysG; = 37, D104N; = 21, P133G; = 25, P133H; = 18, R260A; = 18, R261A, = 15, D262N) was used to show which were statistically impacted by growth on M9 media. All were different than one another with a p value of < 0.001 (see Supplementary Table 1 for details) except those marked here. “*”≤ 0.5 and statistically indistinguishable. There was a significant effect of the CysG variant with an F value of 168.5 and 6 degrees of freedom. Error bars show standard deviations. b ANCOVA analysis of the impact of increasing cobalt concentration on colony size for CysG-deficient E. coli expressing each variant, interpreted as the slope of the line shown in each graph, followed by ANOVA analysis of the slopes to assess whether each variant was affected in a similar or different way. There was a significant interaction between each CysG variant and cobalt concentration with an F value of 43.1 and 6 degrees of freedom (number of colonies counted for each Co2+ concentration provided in Supplementary Table 2). Three groups arose: those that were affected like CysG (D104N and R261A), those that were dramatically affected (P133G and D262N), and those that were more modestly affected (P133H and R260A). Error bars show standard deviations. Source Data are provided as Source Data File SourceData_co-competition.txt.
Fig. 5
Fig. 5. Docking of NAD(H).
a Computational docking of the NAD+ (dark gray) to position the nicotinamide ring close to C15 in precorrin-2 (gold) required removing D81’s side chain and reorienting M172, which take on positions that are antithetical to NAD+ binding when the tetrapyrrole is present in the current crystal structure. The best binding pose for NAD+ is shown after those changes were made, demonstrating how the phosphates must make close approach for the nicotinamide ring to position itself and suggesting that some small conformational changes involving D81 and M172 may occur in solution. b Including D81 and M172 pushes the nicotinamide ring of the NADH (blue) away from the more conjugated sirohydrochlorin (gray) in the docked structure, perhaps explaining how the reaction proceeds after catalysis.
Fig. 6
Fig. 6. P133 and D104 are positioned below and above the sirohydrochlorin.
a Sirohydrochlorin (dark gray) sits direction on top of P133 and below a single water (red sphere). b Upon sirohydrochlorin (dark gray) binding, the DAPK loop flips to position D104 directly over the pyrrole nitrogens, tightly coordinating the water molecule and P106, capping the tetrapyrrole. The apo position of the loop containing D104 (apo-D104) and P106 (apo-P106) is in light gray, whereas the sirohydrochlorin-bound loop containing D104 (shc-D104) and P106 (shc-P106) is in light green.
Fig. 7
Fig. 7. CysGʼs bifunctional dehydrogenase/chelatase active site.
Model for the evolving sirohydrochlorin dehydrogenase product to chelatase substrate, on to the final siroheme product.

Similar articles

Cited by

References

    1. Murphy MJ, Siegel LM. Siroheme and sirohydrochlorin. The basis for a new type of porphyrin-related prosthetic group common to both assimilatory and dissimilatory sulfite reductases. J. Biol. Chem. 1973;248:6911–6919. - PubMed
    1. Peck HD., Jr. Enzymatic basis for assimilatory and dissimilatory sulfate reduction. J. Bacteriol. 1961;82:933–939. doi: 10.1128/JB.82.6.933-939.1961. - DOI - PMC - PubMed
    1. Bali S, et al. Molecular hijacking of siroheme for the synthesis of heme and d1 heme. Proc. Natl. Acad. Sci. USA. 2011;108:18260–18265. doi: 10.1073/pnas.1108228108. - DOI - PMC - PubMed
    1. Bali S, Palmer DJ, Schroeder S, Ferguson SJ, Warren MJ. Recent advances in the biosynthesis of modified tetrapyrroles: the discovery of an alternative pathway for the formation of heme and heme d 1. Cell Mol. Life Sci. 2014;71:2837–2863. doi: 10.1007/s00018-014-1563-x. - DOI - PMC - PubMed
    1. Murphy MJ, Siegel LM, Kamin H, Rosenthal D. Reduced nicotinamide adenine dinucleotide phosphate-sulfite reductase of enterobacteria. II. Identification of a new class of heme prosthetic group: an iron-tetrahydroporphyrin (isobacteriochlorin type) with eight carboxylic acid groups. J. Biol. Chem. 1973;248:2801–2814. - PubMed

Publication types

MeSH terms