Funktion und Struktur des neuen Hämbindeproteins HbpS aus Streptomyceten
Mikrobielle Ökologie und Angewandte Mikrobiologie
Zusammenfassung der Projektergebnisse
We have discovered the Streptomyces three-component system HbpS-SenS-SenR in which the extracellular protein HbpS has an octameric assembly and modulates the activity of the sensor kinase SenS. Having purified the wild-type HbpS and a designed mutant proteins, we obtained high resolution 3D crystal structures showing an octameric assembly of HbpS. Analyses of HbpS-heme binding kinetics support the role HbpS as a heme-sensor and suggested a role in heme transport. Sequence comparisons suggested that HbpS may bind a cobalamin. UV-visible spectroscopy confirmed binding to aquo-cobalamin, but not to other cobalamins. Additional mutational studies allowed the identification of the histidine residue interacting with this compound. HbpS and corresponding close and distant homologues (>10000x) comprise the DUF336 domain either alone or in fusion with other protein domain(s), i.e. PduO. This is a two-domain protein, involved in 1,2-propanediol utilization in the pathogenic Gram-negative bacterium Salmonella enterica, is an ATP:Cob(I)alamin adenosyltransferase but this is a function of the N-terminal domain alone. The role of its C-terminal domain (PduOC) was until now unknown. We showed that PduOC binds heme in vivo. The structure of PduOC co-crystallised with heme was solved (1.9 Å resolution) showing an octameric assembly with four heme moieities. The four heme groups are highly solvent-exposed and the heme iron is hexacoordinated with bis-His ligation by histidines from different monomers. Static light scattering confirmed the octameric assembly in solution, but a mutation of the heme-coordinating histidine caused dissociation into dimers. Isothermal titration calorimetry using the PduOC apoprotein showed strong heme binding (Kd = 1.6 × 10^-7 M). Additional, physiological studies suggest that PduOC:heme plays an important role in the set of cobalamin transformations required for effective catabolism of 1,2-propanediol. The HbpS protein family includes redox-active proteins that interact with the tetrapyrroles heme and cobalamin. Are these protein able to interact with other pyrrole-based secondary metabolites? Are these interactions part of novel signalling networks? Finding of answers these questions will be highly relevant for Streptomycetes as well as for other microorganisms.
Projektbezogene Publikationen (Auswahl)
-
(2014) The extracellular heme-binding protein HbpS from the soil bacterium Streptomyces reticuli is an aquo-cobalamin binder. J Biol Chem 289: 34214-28
Ortiz de Orué Lucana, Darío; Fedosov, Sergey N.; Wedderhoff, Ina; Che, Edith N. & Torda, Andrew E.
-
(2016) Complete genome sequence of Streptomyces reticuli, an efficient degrader of crystalline cellulose. J Biotechnol.
Wibberg, Daniel; Al-Dilaimi, Arwa; Busche, Tobias; Wedderhoff, Ina; Schrempf, Hildgund; Kalinowski, Jörn & Ortiz de Orué Lucana, Darío
-
(2016) Deciphering the transcriptional response mediated by the redox-sensing system HbpS-SenS-SenR from Streptomycetes. PLoS One
Busche, Tobias; Winkler, Anika; Wedderhoff, Ina; Rückert, Christian; Kalinowski, Jörn & Ortiz de Orué Lucana, Darío
-
(2016) The Crystal Structure of the C-terminal domain of the PduO protein from Salmonella enterica: An old fold with a new heme-binding mode. Front Microbiol
Ortiz de Orué Lucana, Darío; Hickey, Neal; Hensel, Michael; Klare, Johann P.; Geremia, Silvano; Tiufiakova, Tatiana & Torda, Andrew E.
