A low pKa cysteine at the active site of mouse methionine sulfoxide reductase A

Methionine sulfoxide reductase A is an essential enzyme in the antioxidant system which scavenges reactive oxygen species through cyclic oxidation and reduction of methionine and methionine sulfoxide. Recently it has also been shown to catalyze the reverse reaction, oxidizing methionine residues to methionine sulfoxide. A cysteine at the active site of the enzyme is essential for both reductase and oxidase activities. This cysteine has been reported to have a pK(a) of 9.5 in the absence of substrate, decreasing to 5.7 upon binding of substrate. Using three independent methods, we show that the pK(a) of the active site cysteine of mouse methionine sulfoxide reductase is 7.2 even in the absence of substrate. The primary mechanism by which the pK(a) is lowered is hydrogen bonding of the active site Cys-72 to protonated Glu-115. The low pK(a) renders the active site cysteine susceptible to oxidation to sulfenic acid by micromolar concentrations of hydrogen peroxide. This characteristic supports a role for methionine sulfoxide reductase in redox signaling.     

Electron Transfer Pathways and Dynamics of Chloroplast NADPH-dependent Thioredoxin Reductase C (NTRC)

NADPH-dependent thioredoxin reductases (NTRs) contain a flavin cofactor and a disulfide as redox-active groups. The catalytic mechanism of standard NTR involves a large conformational change between two configurations. Oxygenic photosynthetic organisms possess a plastid-localized NTR, called NTRC, with a thioredoxin module fused at the C terminus. NTRC is an efficient reductant of 2-Cys peroxiredoxins (2-Cys Prxs) and thus is involved in the protection against oxidative stress, among other functions. Although the mechanism of electron transfer of canonical NTRs is well established, it is not yet known in NTRC. By employing stopped-flow spectroscopy, we have carried out a comparative kinetic study of the electron transfer reactions involving NTRC, the truncated NTR module of NTRC, and NTRB, a canonical plant NTR. Whereas the three NTRs maintain the conformational change associated with the reductive cycle of catalysis, NTRC intramolecular electron transfer to the thioredoxin module presents two kinetic components (k_ET of ∼2 and 0.1 s⁻¹), indicating the occurrence of additional dynamic motions. Moreover, the dynamic features associated with the electron transfer to the thioredoxin module are altered in the presence of 2-Cys Prx. NTRC shows structural constraints that may locate the thioredoxin module in positions with different efficiencies for electron transfer, the presence of 2-Cys Prx shifting the conformational equilibrium of the thioredoxin module to a specific position, which is not the most efficient.     

Structure and Mechanism of Sanguinarine Reductase,an Enzyme of Alkaloid Detoxification

Sanguinarine reductase is a plant enzyme that prevents the cytotoxic effects of benzophenanthridine alkaloids, which are the main phytoalexins of Papaveraceae. The enzyme catalyzes the reduction of sanguinarine, the most toxic benzophenanthridine, which re-enters the cytoplasm after its primary accumulation in the cell wall region has reached a threshold concentration. We present the sequence of the gene and protein of sanguinarine reductase isolated from cell cultures of Eschscholzia californica. High sequence similarities indicate that the enzyme evolved from a plant-specific branch of the ubiquitous Rossmann fold NAD(P)H/NAD(P)⁺ binding reductases, with NADP-dependent epimerases or hydroxysteroid reductases as the most likely ancestors. Based on the x-ray structure of a close homolog, a three-dimensional model of the spatial conformation and catalytic site of sanguinarine reductase was established and used for in silico screening of known three-dimensional structures. Surprisingly, the enzyme shares high structural similarity with enzymes of human and bacterial origin, which have similar functions as the plant homologs but bear little amino acid sequence similarity. Using site-directed mutagenesis, a series of recombinant enzymes was generated and assayed to reveal the impact of individual amino acids and peptides in the catalytic process. It appears that relatively few innovations were required to generate this selective catalyst for alkaloid detoxication, notably an insertion of 13 amino acids and the generation of a novel catalytic triad of Cys-Asp-His were sufficient.     

A “push-pull-restrain” strategy to improve citronellol production in Saccharomyces cerevisiae

Microbial production of monoterpenes has attracted increasing attention in recent years. Up to date, there are only few reports on the biosynthesis of the monoterpene alcohol citronellol that is widely used as fragrant and pharmaceutical intermediates. Here, we engineered Saccharomyces cerevisiae by employing a “push-pull-restrain” strategy to improve citronellol production based on the reduction of geraniol. Starting from a engineered geraniol-producing strain, different reductases were investigated and the best performing iridoid synthase from Catharanthus roseus (CrIS) resulted in 285.89 mg/L enantiomerically pure S-citronellol in shake flasks. Geranyl diphosphate (GPP), the most important precursor for monoterpenes, was enhanced by replacing the wild farnesyl diphosphate synthase (Erg20) with the mutant Erg20^F96W, increasing the citronellol titer to 406.01 mg/L without negative influence on cell growth. Moreover, we employed synthetic protein scaffolds and protein fusion to colocalize four sequential enzymes to achieve better substrate channeling along with the deletion of an intermediate degradation pathway gene ATF1, which elevated the citronellol titer to 972.02 mg/L with the proportion of 97.8% of total monoterpenes in YPD medium. Finally, the engineered strain with complemented auxotrophic markers produced 8.30 g/L of citronellol by fed-batch fermentation, which was the highest citronellol titer reported to date. The multi-level engineering strategies developed here demonstrate the potential of monoterpenes overproduction in yeast, which can serve as a generally applicable platform for overproduction of other monoterpenes.     

Micro-Array Analysis of Resistance for Gemcitabine Results in Increased Expression of Ribonucleotide Reductase Subunits

To study in detail the relation between gene expression and resistance against gemcitabine, a cell line was isolated from a tumor for which gemcitabine resistance was induced in vivo. Similar to the in vivo tumor, resistance in this cell line, C 26-G, was not related to deficiency of deoxycytidine kinase (dCK). Micro-array analysis showed increased expression of ribonucleotide reductase (RR) subunits M1 and M2 as confirmed by real time PCR analysis (28- and 2.7-fold, respectively). In cell culture, moderate cross-resistance (about 2-fold) was observed to 1-ß-D-arabinofuranosylcytosine (ara-C), 2-chloro-2’deoxyadenosine (CdA), LY231514 (ALIMTA), and cisplatin (CDDP), and pronounced cross-resistance (>23-fold) to 2′,2′-difluorodeoxyuridine (dFdU) and 2′,2′-difluorodeoxyguanosine (dFdG). Culture in the absence of gemcitabine reduced resistance as well as RRM1 RNA expression, demonstrating a direct relationship of RRM1 RNA expression with acquired resistance to gemcitabine.     

Pyrrolylbenzothiazole Derivatives as Aldose Reductase Inhibitors

Abstract

2,2-Dimethyl-4-hydroxy-4-androstene-3,17-dione (4) has been synthesized and has been shown to be a powerful competitive inhibitor of aromatase (K_i = 11.4nM). However, compound 4 does not cause time-dependent loss of enzyme activity, in contrast to the unmethylated parent compound, 4-OHA.     

IruO Is a Reductase for Heme Degradation by IsdI and IsdG Proteins in Staphylococcus aureus

Staphylococcus aureus is a common hospital- and community-acquired bacterium that can cause devastating infections and is often multidrug-resistant. Iron acquisition is required by S. aureus during an infection, and iron acquisition pathways are potential targets for therapies. The gene NWMN2274 in S. aureus strain Newman is annotated as an oxidoreductase of the diverse pyridine nucleotide-disulfide oxidoreductase (PNDO) family. We show that NWMN2274 is an electron donor to IsdG and IsdI catalyzing the degradation of heme, and we have renamed this protein IruO. Recombinant IruO is a FAD-containing NADPH-dependent reductase. In the presence of NADPH and IruO, either IsdI or IsdG degraded bound heme 10-fold more rapidly than with the chemical reductant ascorbic acid. Varying IsdI-heme substrate and monitoring loss of the heme Soret band gave a K_m of 15 ± 4 μm, a k_cat of 5.2 ± 0.7 min⁻¹, and a k_cat/K_m of 5.8 × 10³ m⁻¹ s⁻¹. From HPLC and electronic spectra, the major heme degradation products are 5-oxo-δ-bilirubin and 15-oxo-β-bilirubin (staphylobilins), as observed with ascorbic acid. Although heme degradation by IsdI or IsdG can occur in the presence of H₂O₂, the addition of catalase and superoxide dismutase did not disrupt NADPH/IruO heme degradation reactions. The degree of electron coupling between IruO and IsdI or IsdG remains to be determined. Homologs of IruO were identified by sequence similarity in the genomes of Gram-positive bacteria that possess IsdG-family heme oxygenases. A phylogeny of these homologs identifies a distinct clade of pyridine nucleotide-disulfide oxidoreductases likely involved in iron uptake systems. IruO is the likely in vivo reductant required for heme degradation by S. aureus.     

Resistance to AFN-1252 Arises from Missense Mutations in Staphylococcus aureus Enoyl-acyl Carrier Protein Reductase (FabI)

AFN-1252 is a potent antibiotic against Staphylococcus aureus that targets the enoyl-acyl carrier protein reductase (FabI). A thorough screen for AFN-1252-resistant strains was undertaken to identify the spectrum of mechanisms for acquired resistance. A missense mutation in fabI predicted to encode FabI(M99T) was isolated 49 times, and a single isolate was predicted to encode FabI(Y147H). AFN-1252 only bound to the NADPH form of FabI, and the close interactions between the drug and Met-99 and Tyr-147 explained how the mutations would result in resistant enzymes. The clone expressing FabI(Y147H) had a pronounced growth defect that was rescued by exogenous fatty acid supplementation, and the purified protein had less than 5% of the enzymatic activity of FabI. FabI(Y147F) was also catalytically defective but retained its sensitivity to AFN-1252, illustrating the importance of the conserved Tyr-147 hydroxyl group in FabI function. The strains expressing FabI(M99T) exhibited normal growth, and the biochemical properties of the purified protein were indistinguishable from those of FabI. The AFN-1252 K_i^app increased from 4 nm in FabI to 69 nm in FabI(M99T), accounting for the increased resistance of the corresponding mutant strain. The low activity of FabI(Y147H) precluded an accurate K_i measurement. The strain expressing FabI(Y147H) was also resistant to triclosan; however, the strain expressing FabI(M99T) was more susceptible. Strains with higher levels of AFN-1252 resistance were not obtained. The AFN-1252-resistant strains remained sensitive to submicromolar concentrations of AFN-1252, which blocked growth through inhibition of fatty acid biosynthesis at the FabI step.     

Therapeutic Potential of Melatonin Ligands

Melatonin is a neurohormone that is believed to be involved in a wide range of physiological functions. In humans, appropriate clinical trials confirm the efficacy of melatonin or melatoninergic agonists for the MT₁ and MT₂ receptor subtypes in circadian rhythm sleep disorders only. Nevertheless, preclinical animal model studies relevant to human pathologies involving validated reference compounds lead to other therapeutic possibilities. Among these is a recently developed treatment concept for depression, which has been validated by the clinical efficacy of agomelatine, an agent having both MT₁ and MT₂ agonist and 5‐HT_2C antagonist activity. A third melatonin binding site has been purified and characterized as the enzyme quinone reductase 2 (QR2). The physiological role of this enzyme is not yet known. Recent results obtained by different groups suggest: (1) that inhibition of QR2 may lead to “protective” effects and (2) that over‐expression of this enzyme may have deleterious effects. The inhibitory effect of melatonin on QR2 observed in vitro may explain the protective effects reported for melatonin in different animal models, such as cardiac or renal ischemia—effects that have been attributed to the controversial antioxidant properties of the hormone. The development of specific ligands for each of these melatonin binding sites is necessary to link physiological and/or therapeutic effects.