Stepwise Reversion of Multiply Mutated Recombinant Antitrypsin Reveals a Selective Inhibitor of Coagulation Factor XIa as Active as the M358R Variant

Alpha-1 antitrypsin (AAT, also known as alpha-1 proteinase inhibitor or SERPINA1) is the most abundant member of the serpin superfamily found in human plasma. The naturally occurring variant AAT M358R, altered at the P1 position of the critical reactive center loop (RCL), is re-directed away from inhibition of AAT's chief natural target, neutrophil elastase, and toward accelerated inhibition of thrombin (FIIa), kallikrein (Kal), and other proteases such as factor XIa (FXIa). FXIa is an emerging target for the development of antithrombotic agents, since patients with FXI deficiency are protected from thromboembolic disease and do not exhibit a strong bleeding tendency. Previously, we used phage display, bacterial lysate screening, and combinatorial mutagenesis to identify AAT-RC, an engineered AAT M358R with additional changes between RCL positions P7-P3', CLEVEPR-STE [with changes bolded and the P1-P1' (R358-S359) reactive center shown as R-S]. AAT-RC was 279- and 16-fold more selective for FXIa/IIa or FXIa/Kal than AAT M358R; the increased selectivity came at a cost of a 2.3-fold decrease in the rate of FXIa inhibition and a 3.3-fold increase in the stoichiometry of inhibition (SI). Here, we asked which alterations in AAT-RC were most important for the observed increases in selectivity for FXIa inhibition. We back-mutated AAT-RC to AAT-RC-1 (P7-P3' FLEVEPRSTE), AAT-RC-2 (P7-P3' FLEAEPRSTE), and AAT RC-3 (P7-P3' FLEAIPR-STE). Proteins were expressed as cleavable, hexahistidine-tagged glutathione sulfotransferase fusion proteins in E. coli and purified by proteolytic elution from glutathione agarose, with polishing on nickel chelate agarose. Selectivity for FXIa over Kal of AAT-RC-1, −2, and −3 was 14, 21, and 2.3, respectively. AAT-RC-2 inhibited FXIa 31% more rapidly than AAT M358R, with the same SI, and enhanced selectivity for FXIa over Kal, FXa, FXIIa, activated protein C, and FIIa of 25-, 130-, 420-, 440-, and 470-fold, respectively. Structural modeling of the AAT-RC-2/FXIa encounter complex suggested that both E (Glu) substitutions at P3 and P3' may promote FXIa binding via hydrogen bonding to K192 in FXIa. AAT-RC-2 is the most selective and active AAT variant reported to date for FXIa inhibition and will be tested in animal models of thrombosis and bleeding.

High-level expression and molecular characterization of a recombinant prolidase fromEscherichia coliNovaBlue

Long-term use of organophosphorus (OP) compounds has become an increasing global problem and a major threat to sustainability and human health. Prolidase is a proline-specific metallopeptidase that can offer an efficient option for the degradation of OP compounds. In this study, a full-length gene fromEscherichia coliNovaBlue encoding a prolidase (EcPepQ) was amplified and cloned into the commercially-available vector pQE-30 to yield pQE-EcPepQ. The overexpressed enzyme was purified from the cell-free extract of isopropyl thio-β-D-galactoside IPTG-inducedE. coliM15 (pQE-EcPepQ) cells by nickel-chelate chromatography. The molecular mass ofEcPepQ was determined to be about 57 kDa by 12% sodium dodecyl sulfate–polyacrylamide gel electrophoresis and the result of size-exclusion chromatography demonstrated that the enzyme was mainly present in 25 mM Tris–HCl buffer (pH 8.0) as a dimeric form. The optimal conditions forEcPepQ activity were 60 °C, pH 8.0, and 0.1 mM Mn2+ion. Kinetic analysis with Ala-Pro as the substrate showed that theKmandkcatvalues ofEcPepQ were 8.8 mM and 926.5 ± 2.0 s−1, respectively. The thermal unfolding ofEcPepQ followed a two-state process with one well-defined unfolding transition of 64.2 °C. Analysis of guanidine hydrochloride (GdnHCl)-induced denaturation by tryptophan emission fluorescence spectroscopy revealed that the enzyme had a [GdnHCl]0.5,N-Uvalue of 1.98 M. The purified enzyme also exhibited some degree of tolerance to various water/organic co-solvents. Isopropanol and tetrahydrofuran were very detrimental to the enzymatic activity ofEcPepQ; however, other more hydrophilic co-solvents, such as formamide, methanol, and ethylene glycol, were better tolerated. Eventually, the non-negative influence of some co-solvents on both catalytic activity and structural stability ofEcPepQ allows to adjust the reaction conditions more suitable forEcPepQ-catalyzed bioprocess.

Overexpression and Characterization of the Gene Encoding 1,3-Propanediol Oxidoreductase of Citrobacter freundii

Glycerol can be biologically converted to 1,3-propanediol (1,3-PD), a key raw material required for the synthesis of polytrimethylene terephthalate and other polyester fibers. 1,3-propanediol oxidoreductase (PDOR) is the rate-limiting enzyme in 1,3-PD synthesis biologically pathway. In present study, the dhaT gene encoding PDOR was cloned from Citrobacter freundii and overexpressed in E. coli BL21. The recombinant enzyme was purified by nickel-chelate chromatography combined with gel filtration to study the enzymatic characterization. Specific activity of recombinant PDOR was 55.2 U/mg. Km and Vmax values were 8.9 mM, 40.2 U/mg respectively. Holoenzyme of PDOR maybe a decamer of which a monomer has a molecular mass of 43 kDa. This research may form a basis for the future work on biological synthesis of 1, 3-PD.