Co‐purification of nitrate reductase 1 with components of the cytochrome bcc‐aa₃ oxidase supercomplex from spores of Streptomyces coelicolor A3(2)

Abstract

In order to reduce nitrate in vivo, the spore‐specific respiratory nitrate reductase, Nar1, of Streptomyces coelicolor relies on an active cytochrome bcc‐aa3 oxidase supercomplex (bcc‐aa3 supercomplex). This suggests that membrane‐associated Nar1, comprising NarG1, NarH1, and NarI1 subunits, might not act as a classical menaquinol oxidase but could either receive electrons from the bcc‐aa3 supercomplex, or require the supercomplex to stabilize the reductase in the membrane to allow it to function. To address the biochemical basis for this dependence on the bcc‐aa3 supercomplex, we purified two different Strep‐tagged variants of Nar1 and enriched the native enzyme complex from spore extracts using different chromatographic and electrophoretic procedures. Polypeptides associated with the isolated Nar1 complexes were identified using mass spectrometry and included components of the bcc‐aa3 supercomplex, along with an alternative, spore‐specific cytochrome b component, QcrB3. Surprisingly, we also co‐enriched the Nar3 enzyme with Nar1 from the wild‐type strain of S. coelicolor. Two differentially migrating active Nar1 complexes could be identified after clear native polyacrylamide gel electrophoresis; these had masses of approximately 450 and 250 kDa. The distribution of active Nar1 in these complexes was influenced by the presence of cytochrome bd oxidase and by QcrB3; the presence of the latter shifted Nar1 into the larger complex. Together, these data suggest that several respiratory complexes can associate in the spore membrane, including Nar1, Nar3, and the bcc‐aa3 supercomplex. Moreover, these findings provide initial support for the hypothesis that Nar1 and the bcc‐aa3 supercomplex physically associate.

Differences at Species Level and in Repertoires of Secondary Metabolite Biosynthetic Gene Clusters among Streptomyces coelicolor A3(2) and Type Strains of S. coelicolor and Its Taxonomic Neighbors

“…Hatano et al [1] reclassified strain A3(2) from S. coelicolor to S. violaceoruber; however, this reclassification is not widely known. In addition to many recent reports [45][46][47][48], even GenBank still classifies strain A3(2) as S. coelicolor [49,50]. In this study, we showed that strain A3(2) can be reclassified to S. anthocyanicus based on DNA-DNA relatedness, because the whole-genome sequence of the type strain of S. violaceoruber has not yet been published.…”

σE of Streptomyces coelicolor can function both as a direct activator or repressor of transcription

“…SCO4615 is an integrase that is part of the genomic island that was apparently created by the insertion of the pSLP2 vector from S. lividans 27 , 28 . SCO6532 is part of the sco6532-sco6535 operon encoding four subunits of Nar1, a spore-specific nitrate reductase 29 , 30 . As shown in Fig.…”

Tryptophan mutations in G3BP1 tune the stability of a cellular signaling hub by weakening transient interactions with Caprin1 and USP10

“…In our case, the direct interaction has already been established biochemically and crystallographically using purified components. Recent reports, however, have shown the benefits of affinity chromatography in copurifying protein complexes such as antibody stabilized proteins, RNA–protein complexes, and cytochrome supercomplex ( 57 , 58 , 59 ). The homogeneity of these purified complexes has been confirmed by biophysical methods such as small-angle X-ray scattering and protein crystallography ( 8 , 57 ).…”