Characterization of a broadly specific cadaverine N-hydroxylase involved in desferrioxamine B biosynthesis in Streptomyces sviceus

N-hydroxylating flavin-dependent monooxygenases (FMOs) are involved in the biosynthesis of hydroxamate siderophores, playing a key role in microbial virulence. Herein, we report the first structural and kinetic characterization of a novel alkyl diamine N-hydroxylase DesB from Streptomyces sviceus (SsDesB). This enzyme catalyzes the first committed step in the biosynthesis of desferrioxamine B, a clinical drug used to treat iron overload disorders. X-ray crystal structures of the SsDesB holoenzyme with FAD and the ternary complex with bound NADP+ were solved at 2.86 Å and 2.37 Å resolution, respectively, providing a structural view of the active site environment. SsDesB crystallized as a tetramer and the structure of the individual protomers closely resembles the structures of homologous N-hydroxylating FMOs from Erwinia amylovora (DfoA), Pseudomonas aeruginosa (PvdA), and Aspergillus fumigatus (SidA). Using NADPH oxidation, oxygen consumption, and product formation assays, kinetic parameters were determined for various substrates with SsDesB. SsDesB exhibited typical saturation kinetics with substrate inhibition at high concentrations of NAD(P)H as well as cadaverine. The apparent kcat values for NADPH in steady-state NADPH oxidation and oxygen consumption assays were 0.28 ± 0.01 s-1 and 0.24 ± 0.01 s-1, respectively. However, in product formation assays used to measure the rate of N-hydroxylation, the apparent kcat for NADPH (0.034 ± 0.008 s-1) was almost 10-fold lower under saturating FAD and cadaverine concentrations, reflecting an uncoupled reaction, and the apparent NADPH KM was 33 ± 24 μM. Under saturating FAD and NADPH concentrations, the apparent kcat and KM for cadaverine in Csaky assays were 0.048 ± 0.004 s-1 and 19 ± 9 μM, respectively. SsDesB also N-hydroxylated putrescine, spermidine, and L-lysine substrates but not alkyl (di)amines that were branched or had fewer than four methylene units in an alkyl chain. These data demonstrate that SsDesB has wider substrate scope compared to other well-studied ornithine and lysine N-hydroxylases, making it an amenable biocatalyst for the production of desferrioxamine B, derivatives, and other N-substituted products.

Structural analyses of the Group A flavin-dependent monooxygenase PieE reveal a sliding FAD cofactor conformation bridging OUT and IN conformations

Group A flavin-dependent monooxygenases catalyze the cleavage of the oxygen–oxygen bond of dioxygen, followed by the incorporation of one oxygen atom into the substrate molecule with the aid of NADPH and FAD. These flavoenzymes play an important role in many biological processes, and their most distinct structural feature is the choreographed motions of flavin, which typically adopts two distinct conformations (OUT and IN) to fulfill its function. Notably, these enzymes seem to have evolved a delicate control system to avoid the futile cycle of NADPH oxidation and FAD reduction in the absence of substrate, but the molecular basis of this system remains elusive. Using protein crystallography, size-exclusion chromatography coupled to multi-angle light scattering (SEC-MALS), and small-angle X-ray scattering (SEC-SAXS) and activity assay, we report here a structural and biochemical characterization of PieE, a member of the Group A flavin-dependent monooxygenases involved in the biosynthesis of the antibiotic piericidin A1. This analysis revealed that PieE forms a unique hexamer. Moreover, we found, to the best of our knowledge for the first time, that in addition to the classical OUT and IN conformations, FAD possesses a “sliding” conformation that exists in between the OUT and IN conformations. This observation sheds light on the underlying mechanism of how the signal of substrate binding is transmitted to the FAD-binding site to efficiently initiate NADPH binding and FAD reduction. Our findings bridge a gap currently missing in the orchestrated order of chemical events catalyzed by this important class of enzymes.

Expression of a recombinant, 4’-Phosphopantetheinylated, active M. tuberculosis Fatty acid Synthase I in E. coli

Fatty acid synthase 1 (FAS I) from Mycobacterium. tuberculosis (Mtb) is an essential protein and a promising drug target. FAS I is a multi-functional, multi-domain protein that is organized as a large (1.9 MDa) homohexameric complex. Acyl intermediates produced during fatty acid elongation are attached covalently to an acyl carrier protein (ACP) domain. This domain is activated by the transfer of a 4’-Phosphopantetheine (4’-PP, also termed P-pant) group from CoA to ACP catalyzed by a 4’-PP transferase, termed acyl carrier protein synthase (AcpS). In order to obtain an activated FAS I in E. coli, we transformed E. coli with tagged Mtb fas1 and acpS genes encoded by a separate plasmid.We induced the expression of Mtb FAS I following induction of AcpS expression. FAS I was purified by Strep-Tactin affinity chromatography. Activation of Mtb FAS I was confirmed by the identification of a bound P-pant group on serine at position 1808 by mass spectrometry. The purified FAS I displayed biochemical activity shown by spectrophotometric analysis of NADPH oxidation and by CoA production, using the Ellman reaction. The purified Mtb FAS I forms a hexameric complex shown by negative staining and cryo-EM. Purified hexameric and active Mtb FAS I is required for binding and drug inhibition studies and for structurefunction analysis of this enzyme. This relatively simple and short procedure for Mtb FAS I production should facilitate studies of this enzyme.

Characteristic of no-synthase of peripheral blood lymphocytes of patients with rheumatic pathology

It is known that NO is a ubiquitous mediator which acts as a universal modulator of various functions in organism and is produced by three isoforms of NO synthase. Nowadays the role of NO in the development of autoimmune diseases is actively studied. However, it remains unclear the biochemical and biophysical mechanisms of disturbances of NOS activity in blood lymphocytes at autoimmune process. The aim of present work is to study the kinetic properties of NO-synthase of peripheral blood lymphocytes of patients with rheumatic pathology. The study was carried out on peripheral blood lymphocytes isolated from patients with rheumatoid arthritis and ankylosing spondylitis. NOS activity was determined on the saponin-permeabilized blood lymphocytes. The difference between the values of NADPH oxidation with L-Arg and with inhibitor L-NAME reflects the value of the NADPH oxidation, ie total NOS activity. The kinetic properties of NO-synthase in peripheral blood lymphocytes of patients with rheumatic pathology were studied. It was found that the development of rheumatic pathology is associated with an imbalance in the NO synthesis and changes of kinetic parameters of NOS. It was shown that reduction in eNOS activity is accompanied by a sharp increase in activity of its inducible form. It was established that inhibition of eNOS occurs by noncompetitive type. NO production in lymphocytes of patients with rheumatic diseases is mainly realized by iNOS, whereas under normal physiological conditions endothelial form of the enzyme is being involved.

Dehydroepiandrosterone promotes pulmonary artery relaxation by NADPH oxidation-elicited subunit dimerization of protein kinase G 1α

The activity of glucose-6-phosphate dehydrogenase (G6PD) controls a vascular smooth muscle relaxing mechanism promoted by the oxidation of cytosolic NADPH, which has been associated with activation of the 1α form of protein kinase G (PKG-1α) by a thiol oxidation-elicited subunit dimerization. This PKG-1α-activation mechanism appears to contribute to responses of isolated endothelium-removed bovine pulmonary arteries (BPA) elicited by peroxide, cytosolic NADPH oxidation resulting from G6PD inhibition, and hypoxia. Dehydroepiandrosterone (DHEA) is a steroid hormone with pulmonary vasodilator activity, which has beneficial effects in treating pulmonary hypertension. Because multiple mechanisms have been suggested for the vascular effects of DHEA and one of the known actions of DHEA is inhibiting G6PD, we investigated whether it promoted relaxation associated with NADPH oxidation, PKG-1α dimerization, and PKG activation detected by increased vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Relaxation of BPA to DHEA under aerobic or hypoxic conditions was associated with NADPH oxidation, PKG-1α dimerization, and increased VASP phosphorylation. The vasodilator activity of DHEA was markedly attenuated in pulmonary arteries and aorta from a PKG knockin mouse containing a serine in place of a cysteine involved in PKG dimerization. DHEA promoted increased PKG dimerization in lungs from wild-type mice, which was not detected in the PKG knockin mouse model. Thus PKG-1α dimerization is a major contributing factor to the vasodilator actions of DHEA and perhaps its beneficial effects in treating pulmonary hypertension.

Cytoplasmic superoxide dismutase activity is a sensitive indicator of the antioxidant status of the rat liver and brain

Several parameters of the cytoplasmic enzymatic antioxidant system of the liver and brain of the rat have been investigated under conditions of immobilization stress and of an antioxidant preparation in the diet of animals. These included superoxide dismutase (SOD) and glutathione reductase (GR) activities and nonspecific NADPH oxidation. Only changes in the activity of SOD both in the liver and brain were revealed. In the liver of animals that receive no preparation, a decrease in the activity of SOD after 30-min immobilization and its restoration after a 360-min immobilization were observed. In the brain, the activity of SOD decreased only in preconditioned animals after 30 and 360 min of exposure to stress. In addition, the activity of SOD in the brain of preconditioned animals, both stressed and unstressed, was lower than in the corresponding groups of control animals. It is probable that, under the conditions of immobilization stress, the level of reactive oxygen species (ROS) and as a consequence the activity of SOD decrease. The intake of an antioxidant preparation under these conditions seems to be not correct.

Roles for cytosolic NADPH redox in regulating pulmonary artery relaxation by thiol oxidation-elicited subunit dimerization of protein kinase G1α

The activity of glucose-6-phosphate dehydrogenase (G6PD) appears to control a vascular smooth muscle relaxing mechanism regulated through cytosolic NADPH oxidation. Since our recent studies suggest that thiol oxidation-elicited dimerization of the 1α form of protein kinase G (PKG1α) contributes to the relaxation of isolated endothelium-removed bovine pulmonary arteries (BPA) to peroxide and responses to hypoxia, we investigated whether cytosolic NADPH oxidation promoted relaxation by PKG1α dimerization. Relaxation of BPA to G6PD inhibitors 6-aminonicotinamide (6-AN) and epiandrosterone (studied under hypoxia to minimize basal levels of NADPH oxidation and PKG1α dimerization) was associated with increased PKG1α dimerization and PKG-mediated vasodilator-stimulated phosphoprotein (VASP) phosphorylation. Depletion of PKG1α by small inhibitory RNA (siRNA) inhibited relaxation of BPA to 6-AN and attenuated the increase in VASP phosphorylation. Relaxation to 6-AN did not appear to be altered by depletion of soluble guanylate cyclase (sGC). Depletion of G6PD, thioredoxin-1 (Trx-1), and Trx reductase-1 (TrxR-1) in BPA with siRNA increased PKG1α dimerization and VASP phosphorylation and inhibited force generation under aerobic and hypoxic conditions. Depletion of TrxR-1 with siRNA inhibited the effects of 6-AN and enhanced similar responses to peroxide. Peroxiredoxin-1 depletion by siRNA inhibited PKG dimerization to peroxide, but it did not alter PKG dimerization under hypoxia or the stimulation of dimerization by 6-AN. Thus regulation of cytosolic NADPH redox by G6PD appears to control PKG1α dimerization in BPA through its influence on Trx-1 redox regulation by the NADPH dependence of TrxR-1. NADPH regulation of PKG dimerization may contribute to vascular responses to hypoxia that are associated with changes in NADPH redox.