The central step in the assimilation of sulfur is a six-electron reduction of sulfite to sulfide, catalyzed by the oxidoreductase NADPH-dependent assimilatory sulfite reductase (SiR). SiR is composed of two subunits. One is a multidomain flavin binding reductase (SiRFP) and the other an iron-containing oxidase (SiRHP). Both enzymes are primarily globular, as expected from their functions as redox enzymes. Consequently, we know a fair amount about their structures but not how they assemble. Curiously, both structures have conspicuous regions that are structurally undefined, leaving questions about their functions and raising the possibility that they are critical in forming the larger complex. Here, we used ultraviolet-visible and circular dichroism spectroscopy, isothermal titration calorimetry, proteolytic sensitivity tests, electrospray ionization mass spectrometry, and activity assays to explore the effect of altering specific amino acids in SiRFP on their function in the holoenzyme complex. Additionally, we used computational analysis to predict the propensity for intrinsic disorder within both subunits and found that SiRHP's N-terminus is predicted to have properties associated with intrinsic disorder. Both proteins also contained internal regions with properties indicative of intrinsic disorder. We showed that SiRHP's N-terminal disordered region is critical for complex formation. Together with our analysis of SiRFP amino acid variants, we show how molecular interactions outside the core of each SiR globular enzyme drive complex assembly of this prototypical oxidoreductase.

Cardiovascular disease is characterized by aberrant and excessive extracellular matrix (ECM) remodelling, termed fibrosis. Fibrotic remodelling is typically triggered by inflammation, which occurs systemically in obesity. Despite the contribution of fibrosis to adverse clinical outcomes and disease progression, there are no available treatments for this condition. Developing therapeutics for chronic conditions requires an understanding ofin vivoECM regulation, and how the ECM responds to a systemic challenge. We have therefore developed aDrosophilamodel for obesity via chronic high fat diet feeding and evaluated the response of the cardiac ECM to this metabolic challenge. We found that this model displays a striking disorganization of the cardiac ECM, with corresponding deficits in heart function. Our study shows that different genotypes tolerate varying levels of high fat diets, and that some genotypes may require a different percentage of fat supplementation for achieving an optimal obesity phenotype.

In enterobacteria like Escherichia coli, NADPH‐dependent assimilatory Sulfite Reductase (SiR) is the central enzyme in the sulfur assimilation pathway. SiR performs the six‐electron reduction of sulfite (SO3−2) to sulfide (S−2). SiR is a soluble multimeric enzyme with two subunits that combine in a 2:1 ratio to form a stable dodecameric complex. The α subunit (SiRFP) is a cytochrome P450 oxidoreductase (CYPOR) homolog. The β subunit (SiRHP) is a monomeric metalloenzyme with a Fe4S4 cluster and a porphyrinoid group called siroheme that forms the active site for the complex. SiRFP has the prototypical, three‐domain architecture of CYPOR that transfers electrons from the cellular donor NADPH to its FAD and FMN cofactors sequentially, ultimately delivering them to the binding partner, in this case SiRHP. However, SiRFP differs from other CYPORs in several ways: 1) SiRFP is a soluble protein that forms a homo‐octamer when expressed in the absence of SiRHP and 2) SiRFP forms a stable complex with SiRHP. The main aim of our research is to understand the electron transfer between the subunits in the context of the unique stoichiometry and structure of the complex. We identified two regions of SiRFP whose solvent accessibility changes upon assembly with SiRHP. The two regions of the structure are on different faces of the protein: one is in the N‐terminal FMN‐binding domain and the other is in the C‐terminal NADPH binding domain. Electrons are known to pass from the FMN binding domain to SiRHP. Interestingly, the interface that is part of the NADPH binding domain is sufficient to form a tight complex with SiRHP, suggesting the interface within the FMN binding domain is transient. To test this hypothesis, we synthesized three point variants: Y101A, Q191K, and V500D. Y101 is part of the FMN binding domain. Q191 plays a role in the conformational change that is known to govern electron transfer in CYPOR. V500 is part of the SiRFP region that interacts with SiRHP. Then, we used complementation assays and isothermal titration calorimetry (ITC) to probe the effect of these mutations. The three combined mutations has a synergic effect, resulting in cells that were unable to grow in minimal media. The ITC data showed that only the SiRFP V500D and variants combined with it had a reduced affinity for SiRHP respect to the wild‐type enzyme. From these results we conclude that SiR complex formation is driven by hydrophobic interactions in the C‐terminus of SiRFP, far from the site of electron transfer to its SiRHP partner. In contrast, transient interactions involving Y101 are important for electron transfer from the FMN cofactor to the metallic cofactors in SiRHP.Support or Funding InformationMCB1149763