Crystal structure and activity of a de novo enzyme, ferric enterobactin esterase Syn-F4.

Producing novel enzymes that are catalytically active in vitro and biologically functional in vivo is a key goal of synthetic biology. Previously, we reported Syn-F4, the first de novo protein that meets both criteria. Syn-F4 hydrolyzed the siderophore ferric enterobactin, and expression of Syn-F4 allowed an inviable strain of Escherichia coli (Δfes) to grow in iron-limited medium. Here, we describe the crystal structure of Syn-F4. Syn-F4 forms a dimeric 4-helix bundle. Each monomer comprises two long α-helices, and the loops of the Syn-F4 dimer are on the same end of the bundle (syn topology). Interestingly, there is a penetrated hole in the central region of the Syn-F4 structure. Extensive mutagenesis experiments in a previous study showed that five residues (Glu26, His74, Arg77, Lys78, and Arg85) were essential for enzymatic activity in vivo. All these residues are located around the hole in the central region of the Syn-F4 structure, suggesting a putative active site with a catalytic dyad (Glu26-His74). The complete inactivity of purified proteins with mutations at the five residues supports the putative active site and reaction mechanism. Molecular dynamics and docking simulations of the ferric enterobactin siderophore binding to the Syn-F4 structure demonstrate the dynamic property of the putative active site. The structure and active site of Syn-F4 are completely different from native enterobactin esterase enzymes, thereby demonstrating that proteins designed de novo can provide life-sustaining catalytic activities using structures and mechanisms dramatically different from those that arose in nature.

RNA Polymerase: Step-by-Step Kinetics and Mechanism of Transcription Initiation.

To determine the step-by-step kinetics and mechanism of transcription initiation and escape by E. coli RNA polymerase from the λPR promoter, we quantify the accumulation and decay of transient short RNA intermediates on the pathway to promoter escape and full-length (FL) RNA synthesis over a wide range of NTP concentrations by rapid-quench mixing and phosphorimager analysis of gel separations. Experiments are performed at 19 °C, where almost all short RNAs detected are intermediates in FL-RNA synthesis by productive complexes or end-products in nonproductive (stalled) initiation complexes and not from abortive initiation. Analysis of productive-initiation kinetic data yields composite second-order rate constants for all steps of NTP binding and hybrid extension up to the escape point (11-mer). The largest of these rate constants is for incorporation of UTP into the dinucleotide pppApU in a step which does not involve DNA opening or translocation. Subsequent steps, each of which begins with reversible translocation and DNA opening, are slower with rate constants that vary more than 10-fold, interpreted as effects of translocation stress on the translocation equilibrium constant. Rate constants for synthesis of 4- and 5-mer, 7-mer to 9-mer, and 11-mer are particularly small, indicating that RNAP-promoter interactions are disrupted in these steps. These reductions in rate constants are consistent with the previously determined ∼9 kcal cost of escape from λPR. Structural modeling and previous results indicate that the three groups of small rate constants correspond to sequential disruption of in-cleft, -10, and -35 interactions. Parallels to escape by T7 RNAP are discussed.

Coagulation-membrane filtration of Chlorella vulgaris.

Filtration-based separation of Chlorella vulgaris, a species with excellent potential for CO(2) capture and lipid production, was investigated using a surface-modified hydrophilic polytetrafluoroethylene (PTFE) membrane. Coagulation using polyaluminum chloride (PACl) attained maximum turbidity removal at 200 mg L(-1) as Al(2)O(3). The membrane filtration flux at 1 bar increased as the PACl dose increased, regardless of overdosing in the coagulation stage. The filtered cake at the end of filtration tests peaked in solid content at 10 mg L(-1) as Al(2)O(3), reaching 34% w/w, roughly two times that of the original suspension. Differential scanning calorimetry (DSC) tests demonstrate that the cake with minimum water-solid binding strength produced the driest filter cake. Coagulation using 10 mg L(-1) PACl as Al(2)O(3), followed by PTFE membrane filtration at 1 bar, is an effective process for harvesting C. vulgaris from algal froth.

Harvesting of Scenedesmus obliquus FSP-3 using dispersed ozone flotation.

The Scenedesmus obliquus FSP-3, a species with excellent potential for CO(2) capture and lipid production, was harvested using dispersed ozone flotation. While air aeration does not, ozone produces effective solid-liquid separation through flotation. Ozone dose applied for sufficient algal flotation is similar to those used in practical drinking waterworks. The algae removal rate, surface charge, and hydrophobicity of algal cells, and fluorescence characteristics and proteins and polysaccharides contents of algogenic organic matter (AOM) were determined during ozonation. Proteins released from tightly bound AOM are essential to modifying the hydrophobicity of bubble surfaces for easy cell attachment and to forming a top froth layer for collecting floating cells. Humic substances in the suspension scavenge dosed ozone that adversely affects ozone flotation efficiency of algal cells.

Dispersed ozone flotation of Chlorella vulgaris.

Flotation separation of Chlorella vulgaris, a species with excellent potential for CO(2) capture and lipid production, was studied using dispersed ozone gas. Pure oxygen aeration did not yield flotation. Conversely, applying ozone effectively separation algae from broth through flotation. The ozone dose applied for sufficient algal flotation is <0.05 mg/g biomass, much lower than those used in practical drinking waterworks (0.1-0.3 mg/g suspended solids). Main products, lipid C16:0, was effectively collected in the flotage phase. The algae removal rate, surface charge, and hydrophobicity of algal cells, and proteins and polysaccharides contents of algogenic organic matter (AOM) were determined. Certain quantities of proteins were present in the cultivated algal suspension, hence, minimal quantity of ozone was required to release intracellular proteins as surfactants to lead to effective flotation.