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A polyene macrolide antibiotic tetramycin biosynthetic gene cluster was identified by genome mining and isolated from Streptomyces hygrospinosus var. beijingensis. Genetic and in silico analyses gave insights into the mechanism of biosynthesis of tetramycin, and a model of the tetramycin biosynthetic pathway is proposed. Inactivation of a cytochrome P450 monooxygenase gene, tetrK, resulted in the production of a tetramycin B precursor: tetramycin A, which lacks a hydroxy group in its polyol region. TetrK was subsequently overexpressed heterologously in E. coli with a His(6) tag, and purified TetrK efficiently hydroxylated tetramycin A to afford tetramycin B. Kinetic studies revealed no inhibition of TetrK by substrate or product. Surprisingly, sequence-alignment analysis showed that TetrK, as a hydroxylase, has much higher homology with epoxidase PimD than with hydroxylases NysL and AmphL. The 3D structure of TetrK was then constructed by homology modeling with PimD as reference. Although TetrK and PimD catalyzed different chemical reactions, homology modeling indicated that they might share the same catalytic sites, despite also possessing some different sites correlated with substrate binding and substrate specificity. These findings offer good prospects for the production of improved antifungal polyene analogues.

We report a suite of enzyme redesign protocol based on the surface charge-charge interaction calculation, which is potentially applied to improve the stability of an enzyme without compromising its catalytic activity. Together with the experimental validation, we have released a suite of enzyme redesign algorithm Enzyme Thermal Stability System, written based on our model, for open access to meet the needs in wet labs. Lipk107, a lipase of a versatile industrial use, was chosen to test our software. Our calculation determined that four residues, D113, D149, D213, and D253, located on the surface of LipK107 were critical to the stability of the enzyme. The model was validated with mutagenesis at these four residues followed by stability and activity tests. LipK107 mutants D113A and D149K were more resistant to thermal inactivation with~10 C higher halfinactivation temperature than wild-type LipK107. Moreover, mutant D149K exhibited significant retention in residual activity under constant heat, showing a 14-fold increase in the halfinactivation time at 50 C. Activity tests showed that these mutants retained the equal or higher specific activity, among which noteworthy was the mutant D253A with as much as 20% higher Additional Supporting Information may be found in the online version of this article. Published by Wiley-Blackwell. V C 2013 The Protein Society activity. We suggest that our protocol could be used as a general guideline to redesign protein enzymes with increased stabilities and enhanced activities.

Coenzyme engineering, especially for altered coenzyme specificity, has been a research hotspot for more than a decade. In the present study, a novel computational strategy that enhances the hydrogen-bond interaction between an enzyme and a coenzyme was developed and utilized to alter the coenzyme preference. This novel computational strategy only required the structure of the target enzyme. No other homologous enzymes were needed to achieve alteration in the coenzyme preference of a certain enzyme. Using our novel strategy, Gox2181 was reconstructed from exhibiting complete NADPH preference to exhibiting dual cofactor specificity for NADH and NADPH. Structure-guided Gox2181 mutants were designed in silico and molecular dynamics simulations were performed to evaluate the strength of hydrogen-bond interactions between the enzyme and the coenzyme NADPH. Three Gox2181 mutants displaying high structure stability and structural compatibility to NADH/NADPH were chosen for experimental confirmation. Among the three Gox2181 mutants, Gox2181-Q20R&D43S showed the highest enzymatic activity by utilizing NADPH as its coenzyme, which was even better than the wild-type enzyme. In addition, isothermal titration calorimetry analysis further verified that Gox2181-Q20R&D43S was able to interact with NADPH but the wild-type enzyme could not. This novel computational strategy represents an insightful approach for altering the cofactor preference of target enzymes. DatabaseModel data have been deposited in the Protein Model Database database under the accession numbers PM0079165, PM0079166, PM0079167, PM0079168 and PM0079169.

Interleukin-15 (IL-15) can promote both innate and adaptive immune reactions by stimulating CD8+/CD4+ T cells and natural killer cells (NK) while showing no effect in activating T-regulatory (Treg) cells or inducing activation-associated death among effector T cells and NK cells. Thus, IL-15 is considered as one of the most promising molecules for antitumor immune therapy. To improve the drug-like properties of natural IL-15, we create an IL-15-based molecule, named P22339, with the following characteristics: 1) building a complex of IL-15 and the Sushi domain of IL-15 receptor α chain to enhance the agonist activity of IL-15 via transpresentation; 2) through a rational structure-based design, creating a disulfide bond linking the IL-15/Sushi domain complex with an IgG1 Fc to augment its half-life. P22339 demonstrates excellent developability, pharmacokinetic and pharmacodynamic properties as well as antitumor efficacy in both in vitro assessments and in vivo studies. It significantly suppresses tumor growth and metastasis in rodent models, and activates T effector cells and NK cells in cynomolgus monkey. Overall, these data suggest that P22339 has a great potential for cancer immunotherapy.