Structure-Function Analysis of the Bifunctional CcsBA Heme Exporter and Cytochrome c Synthetase.

Although intracellular heme trafficking must occur for heme protein assembly, only a few heme transporters have been unequivocally discovered and nothing is known about their structure or mechanisms. Cytochrome c biogenesis in prokaryotes requires the transport of heme from inside to outside for stereospecific attachment to cytochrome c via two thioether bonds (at CXXCH). The CcsBA integral membrane protein was shown to transport and attach heme (and thus is a cytochrome c synthetase), but the structure and mechanisms underlying these two activities are poorly understood. We employed a new cysteine/heme crosslinking tool that traps endogenous heme in heme binding sites. We combined these data with a comprehensive imidazole correction approach (for heme ligand interrogation) to map heme binding sites. Results illuminate the process of heme transfer through the membrane to an external binding site (called the WWD domain). Using meta-genomic data (GREMLIN) and Rosetta modeling programs, a structural model of the transmembrane (TM) regions in CcsBA were determined. The heme mapping data were then incorporated to model the TM heme binding site (with TM-His1 and TM-His2 as ligands) and the external heme binding WWD domain (with P-His1 and P-His2 as ligands). Other periplasmic structure/function studies facilitated modeling of the full CcsBA protein as a framework for understanding the mechanisms. Mechanisms are proposed for heme transport from TM-His to WWD/P-His and subsequent stereospecific attachment of heme. A ligand exchange of the P-His1 for histidine of CXXCH at the synthetase active site is suggested.IMPORTANCE The movement or trafficking of heme is critical for cellular functions (e.g., oxygen transport and energy production); however, intracellular heme is tightly regulated due to its inherent cytotoxicity. These factors, combined with the transient nature of transport, have resulted in a lack of direct knowledge on the mechanisms of heme binding and trafficking. Here, we used the cytochrome c biogenesis system II pathway as a model to study heme trafficking. System II is composed of two integral membrane proteins (CcsBA) which function to transport heme across the membrane and stereospecifically position it for covalent attachment to apocytochrome c We mapped two heme binding domains in CcsBA and suggest a path for heme trafficking. These data, in combination with metagenomic coevolution data, are used to determine a structural model of CcsBA, leading to increased understanding of the mechanisms for heme transport and the cytochrome c synthetase function of CcsBA.

Structurally Mapping Endogenous Heme in the CcmCDE Membrane Complex for Cytochrome c Biogenesis.

Although many putative heme transporters have been discovered, it has been challenging to prove that these proteins are directly involved with heme trafficking in vivo and to identify their heme binding domains. The prokaryotic pathways for cytochrome c biogenesis, Systems I and II, transport heme from inside the cell to outside for stereochemical attachment to cytochrome c, making them excellent models to study heme trafficking. System I is composed of eight integral membrane proteins (CcmA-H) and is proposed to transport heme via CcmC to an external "WWD" domain for presentation to the membrane-tethered heme chaperone, CcmE. Herein, we develop a new cysteine/heme crosslinking approach to trap and map endogenous heme in CcmC (WWD domain) and CcmE (defining "2-vinyl" and "4-vinyl" pockets for heme). Crosslinking occurs when either of the two vinyl groups of heme localize near a thiol of an engineered cysteine residue. Double crosslinking, whereby both vinyls crosslink to two engineered cysteines, facilitated a more detailed structural mapping of the heme binding sites, including stereospecificity. Using heme crosslinking results, heme ligand identification, and genomic coevolution data, we model the structure of the CcmCDE complex, including the WWD heme binding domain. We conclude that CcmC trafficks heme via its WWD domain and propose the structural basis for stereochemical attachment of heme.

Utilization of inulin-containing waste in industrial fermentations to produce biofuels and bio-based chemicals.

Inulins are polysaccharides that belong to an important class of carbohydrates known as fructans and are used by many plants as a means of storing energy. Inulins contain 20 to several thousand fructose units joined by ß-2,1 glycosidic bonds, typically with a terminal glucose unit. Plants with high concentrations of inulin include: agave, asparagus, coffee, chicory, dahlia, dandelion, garlic, globe artichoke, Jerusalem artichoke, jicama, onion, wild yam, and yacón. To utilize inulin as its carbon and energy source directly, a microorganism requires an extracellular inulinase to hydrolyze the glycosidic bonds to release fermentable monosaccharides. Inulinase is produced by many microorganisms, including species of Aspergillus, Kluyveromyces, Penicillium, and Pseudomonas. We review various inulinase-producing microorganisms and inulin feedstocks with potential for industrial application as well as biotechnological efforts underway to develop sustainable practices for the disposal of residues from processing inulin-containing crops. A multi-stage biorefinery concept is proposed to convert cellulosic and inulin-containing waste produced at crop processing operations to valuable biofuels and bioproducts using Kluyveromyces marxianus, Yarrowia lipolytica, Rhodotorula glutinis, and Saccharomyces cerevisiae as well as thermochemical treatments.

Growth, ethanol production, and inulinase activity on various inulin substrates by mutant Kluyveromyces marxianus strains NRRL Y-50798 and NRRL Y-50799.

Economically important plants contain large amounts of inulin. Disposal of waste resulting from their processing presents environmental issues. Finding microorganisms capable of converting inulin waste to biofuel and valuable co-products at the processing site would have significant economic and environmental impact. We evaluated the ability of two mutant strains of Kluyveromyces marxianus (Km7 and Km8) to utilize inulin for ethanol production. In glucose medium, both strains consumed all glucose and produced 0.40 g ethanol/g glucose at 24 h. In inulin medium, Km7 exhibited maximum colony forming units (CFU)/mL and produced 0.35 g ethanol/g inulin at 24 h, while Km8 showed maximum CFU/mL and produced 0.02 g ethanol/g inulin at 96 h. At 24 h in inulin + glucose medium, Km7 produced 0.40 g ethanol/g (inulin + glucose) and Km8 produced 0.20 g ethanol/g (inulin + glucose) with maximum CFU/mL for Km8 at 72 h, 40 % of that for Km7 at 36 h. Extracellular inulinase activity at 6 h for both Km7 and Km8 was 3.7 International Units (IU)/mL.