The Second-Shell Metal Ligands of Human Arginase Affect Coordination of the Nucleophile and Substrate†

ABSTRACT: The active sites of eukaryotic arginase enzymes are strictly conserved, especially the first- and second-shell ligands that coordinate the two divalent metal cations that generate a hydroxide molecule for nucleophilic attack on the guanidinium carbon of L-arginine and the subsequent production of urea and L-ornithine. Here by using comprehensive pairwise saturation mutagenesis of the first- and second-shell metal ligands in human arginase I, we demonstrate that several metal binding ligands are actually quite tolerant to amino acid substitutions. Of>2800 double mutants of first- and second-shell residues analyzed, we found more than 80 unique amino acid substitutions, of which four were in first-shell residues. Remarkably, certain second-shell mutations could modulate the binding of both the nucleophilic water/hydroxide molecule and substrate or product ligands, resulting in activity greater than that of the wild-type enzyme. The data presented here constitute the first comprehensive saturation mutagenesis analysis of a metallohydrolase active site and reveal that the strict conservation of the second-shell metal binding residues in eukaryotic arginases does not reflect kinetic optimization of the enzyme during the course of evolution. Arginases (EC 3.5.3.1) are typically homotrimeric enzymes with an R/β fold comprising an eight-strand β-sheet surrounded by several helices. The enzyme contains a dinuclear metal center tha

Engineered primate L-methioninase for therapeutic purposes

Methods and compositions relating to the engineering of an improved protein with methionine-γ-lyase enzyme activity are described. For example, in certain aspects there may be disclosed a modified cystathionine-γ-lyase (CGL) comprising one or more amino acid substitutions and capable of degrading methionine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with methionine depletion using the disclosed proteins or nucleic acids.Board of Regents, University of Texas Syste

Engineering and preclinical evaluation of a human enzyme immune checkpoint inhibitor for cancer therapy

The work presented here builds on the findings that the amino acid kynurenine (L-Kyn) synthesized via the enzymes indoleamine/tryptophan dioxygenase (IDO/TDO) mediates cancer immune suppression in a paracrine fashion and evaluates the hypothesis that systemic elimination of secreted Kyn in the extracellular space using an engineered human kynureninase enzyme (KYNase) to degrade it into inactive metabolites will have pronounced anti-tumor activity and overcome the limitations associated with IDO/TDO inhibitors. The significance of L-Kyn production in cancer is well recognized and has led to the development of inhibitors of IDO and TDO; with at least one IDO inhibitor currently undergoing Phase II/III clinical evaluation. However IDO or TDO inhibition is problematic for cancer therapy because: (1) there are two isoforms of IDO and together with TDO, the inhibition of all possible pathways for Kynurenine generation at present requires the generation of multiple small molecule inhibitors; (2) resistance to inhibition can arise, and (3) as a single agent IDO/TDO inhibitors show very little efficacy. Wild-type human KYNase has a strong preference for the degradation of 3’OH Kynurenine (OH-Kyn) with a kcat/KM = 105 M-1s-1, about 1,000 fold higher than that displayed for L-Kyn degradation (102 M-1s-1). In contrast some bacterial enzymes such as the KYNase from P.fluorescens displays a nearly equal and opposite substrate preference for Kyn over OH-Kyn. Although the P.fluorescens KYNase has ideal kinetics desired in an enzyme therapeutic targeting tumor L-Kyn, its high immunogenicity render it unsuitable as a clinical candidate, necessitating the engineering of a human KYNase with the requisite pharmacological properties. Therefore we undertook a directed evolution campaign coupled with high throughput competitive genetic selections and screening strategies to engineer the human KYNase enzyme, creating variants with \u3e 500 fold increases in catalytic activity towards L-Kyn. Engineered human KYNase enzymes were then PEGylated for long circulatory persistence and administered to mice bearing murine cancer allografts and evaluated for efficacy and PK/PD. We found that administration of PEGylated engineered human KYNase enzymes resulted in lowered systemic L-Kyn levels accompanied by significant tumor growth retardation, extended survival and even complete regressions in a manner similar to that observed with immune checkpoint inhibitors such as anti-PD. Flow cytometric analysis showed a significant increase in the proportion of TCRB+ T cells in the tumor-infiltrating lymphocytes (TILs) that is consistent with significant incorporation of BrdU in CD4+ and CD8+ T cells in the tumor-draining lymph nodes (dLNs) and TILs compared to control mice treated with deactivated enzyme. Additionally, marked elevation of CD8+ and CD4+ T cells expressing granzyme (Gzm)B and interferon (IFNG) was observed in the active-enzyme-treated mice, further highlighting the importance of L-Kyn in tumor evasion of immune surveillance. As a monotherapy, small molecule inhibitors of IDO1 display at most marginal anti-cancer activity in animal models as well as in clinical trials likely due to the redundancy of Kyn biosynthetic pathways; necessitating combinations of IDO1/TDO inhibitors. The therapeutic enzyme approach using engineered human KYNase represents an effective “first in class” drug for restoring T-cell immunity for cancer eradication, without the limitations of IDO1/TDO inhibition. This work further demonstrates the utility of “enzymes as drugs” in targeting aberrantly regulated metabolites in disease states

Administration of kynurenine depleting enzymes for tumor therapy

Methods and compositions related to the use of a protein with kynureninase activity are described. For example, in certain aspects there may be disclosed a modified kynureninase capable of degrading kynurenine. Furthermore, certain aspects of the invention provide compositions and methods for the treatment of cancer with kynurenine depletion using the disclosed proteins or nucleic acids.Board of Regents, University of Texas Syste

Promiscuous Partitioning of a Covalent Intermediate Common in the Pentein Superfamily

SummaryMany enzymes in the pentein superfamily use a transient covalent intermediate in their catalytic mechanisms. Here we trap and determine the structure of a stable covalent adduct that mimics this intermediate using a mutant dimethylarginine dimethylaminohydrolase and an alternative substrate. The interactions observed between the enzyme and trapped adduct suggest an altered angle of attack between the nucleophiles of the first and second half-reactions of normal catalysis. The stable covalent adduct is also capable of further reaction. Addition of imidazole rescues the original hydrolytic activity. Notably, addition of other amines instead yields substituted arginine products, which arise from partitioning of the intermediate into the evolutionarily related amidinotransferase reaction pathway. The enzyme provides both selectivity and catalysis for the amidinotransferase reaction, underscoring commonalities among the reaction pathways in this mechanistically diverse enzyme superfamily. The promiscuous partitioning of this intermediate may also help to illuminate the evolutionary history of these enzymes

A non-hydrodynamical model for acceleration of line-driven winds in Active Galactic Nuclei

We present a study of the acceleration phase of line-driven winds in AGNs, in order to examine the physical conditions for the existence of such winds for a wide variety of initial conditions. We built a simple and fast non-hydrodynamic model, QWIND, where we assume that a wind is launched from the accretion disc at supersonic velocities of the order of a few 10^2 km/s and we concentrate on the subsequent supersonic phase. We show that this model can produce a wind with terminal velocities of the order of 10^4 km/s. There are three zones in the wind, only the middle one of which can launch a wind: in the inner zone the wind is too ionized and so experiences only the Compton radiation force which is not effective in accelerating gas. This inner failed wind however plays an important role in shielding the next zone, lowering the ionization parameter there. In the middle zone the lower ionization of the gas leads to a much larger radiation force and the gas achieves escape velocity This middle zone is quite thin (about 100 gravitational radii). The outer, third, zone is shielded from the UV radiation by the central wind zone and so does not achieve a high enough acceleration to reach escape velocity. We also describe a simple analytic approximation of our model, based on neglecting the effects of gravity during the acceleration phase. This analytic approach is in agreement with the results of the numerical code, and is a powerful way to check whether a radiation driven wind can be accelerated with a given set of initial parameters. Our analytical analysis and the fast QWIND model are in agreement with more complex hydrodynamical models, and allow an exploration of the dependence of the wind properties for a wide set of initial parameters: black hole mass, Eddington ratio, initial density profile, X-ray to UV ratio.Comment: 15 pages, 9 figures. Accepted for publication in Astronomy & Astrophysic

Curbside Recycling in the U.S.a.: Convenience and Mandatory Participation

This research examines the relationship between the success of a residential curbside recycling program (RCRP), measured as material recovery rate (MRR), and two program factors: (1) whether or not participation is mandated; and (2) convenience, measured by container provision, collection frequency and collection day relative to municipal solid waste collection day. Residential curbside recycling programs, with correct strategies and program design, can be an important part of solid waste management plans world-wide. While residential curbside recycling programs are growing in popularity, many basic design questions lie unanswered and successful program strategies are not always obvious. Data from 357 residential curbside recycling programs in the United States are used to test the hypotheses. Mandatory participation residential curbside recycling programs are seen to collect more material than voluntary participation residential curbside recycling programs. Container provision appears effective for voluntary, but not mandatory, residential curbside recycling programs. Increasing collection frequency appears to have a small positive effect on residential curbside recycling program success, while collection day has little effect on material recovery rate.Yeshttps://us.sagepub.com/en-us/nam/manuscript-submission-guideline

A Search for Dark Higgs Bosons

Recent astrophysical and terrestrial experiments have motivated the proposal of a dark sector with GeV-scale gauge boson force carriers and new Higgs bosons. We present a search for a dark Higgs boson using 516 fb-1 of data collected with the BABAR detector. We do not observe a significant signal and we set 90% confidence level upper limits on the product of the Standard Model-dark sector mixing angle and the dark sector coupling constant.Comment: 7 pages, 5 postscript figures, published version with improved plots for b/w printin

Search for the standard model Higgs boson in the H to ZZ to 2l 2nu channel in pp collisions at sqrt(s) = 7 TeV

A search for the standard model Higgs boson in the H to ZZ to 2l 2nu decay channel, where l = e or mu, in pp collisions at a center-of-mass energy of 7 TeV is presented. The data were collected at the LHC, with the CMS detector, and correspond to an integrated luminosity of 4.6 inverse femtobarns. No significant excess is observed above the background expectation, and upper limits are set on the Higgs boson production cross section. The presence of the standard model Higgs boson with a mass in the 270-440 GeV range is excluded at 95% confidence level.Comment: Submitted to JHE