Exhibited Substrate Inhibition Research Articles

Mapping roles of active site residues in the acceptor site of the PA3944 Gcn5-related N-acetyltransferase enzyme.

An increased understanding of how the acceptor site in Gcn5-related N-acetyltransferase (GNAT) enzymes recognizes various substrates provides important clues for GNAT functional annotation and their use as chemical tools. In this study, we explored how the PA3944 enzyme from Pseudomonas aeruginosa recognizes three different acceptor substrates, including aspartame, NANMO, and polymyxin B, and identified acceptor residues that are critical for substrate specificity. To achieve this, we performed a series of molecular docking simulations and tested methods to identify acceptor substrate binding modes that are catalytically relevant. We found that traditional selection of best docking poses by lowest S scores did not reveal acceptor substrate binding modes that were generally close enough to the donor for productive acetylation. Instead, sorting poses based on distance between the acceptor amine nitrogen atom and donor carbonyl carbon atom placed these acceptor substrates near residues that contribute to substrate specificity and catalysis. To assess whether these residues are indeed contributors to substrate specificity, we mutated seven amino acid residues to alanine and determined their kinetic parameters. We identified several residues that improved the apparent affinity and catalytic efficiency of PA3944, especially for NANMO and/or polymyxin B. Additionally, one mutant (R106A) exhibited substrate inhibition toward NANMO, and we propose scenarios for the cause of this inhibition based on additional substrate docking studies with R106A. Ultimately, we propose that this residue is a key gatekeeper between the acceptor and donor sites by restricting and orienting the acceptor substrate within the acceptor site.

Electron withdrawing group-dependent substrate inhibition of an α-ketoamide reductase from Saccharomyces cerevisiae

Aldo-keto reductases remain enzymes of interest in biocatalysis due to their ability to reduce carbonyls to alcohols stereospecifically. Based on genomic sequence, we identified aldo-keto reductases of a S. cerevisiae strain extracted from an ancient amber sample. One of the putative enzymes, AKR 163, displays 99% identity with α-amide ketoreductases from the S288C and YJM248 S. cerevisiae strains, which have been investigated for biocatalytic applications. To further investigate AKR 163, we successfully cloned, expressed in E.coli as a glutathione-S-transferase fusion protein, and affinity purified AKR 163. Kinetic studies revealed that AKR 163 experiences strong substrate inhibition by substrates containing halogen atoms or other electron withdrawing groups adjacent to the reactive carbonyl, with Ki values ranging from 0.29 to 0.6 mM and KM values ranging from 0.38 to 0.9 mM at pH 8.0. Substrates without electron withdrawing groups do not display substrate inhibition kinetics and possess much larger KM values between 83 and 260 mM under the same conditions. The kcat values ranged from 0.5 to 2.5s−1 for substrates exhibiting substrate inhibition and 0.22 to 0.52s−1 for substrates that do not engage in substrate inhibition. Overall, the results are consistent with rate-limiting dissociation of the NADP+ cofactor after hydride transfer when electron withdrawing groups are present and activating the reduction step. This process leads to a buildup of enzyme-NADP+ complex that is susceptible to binding and inhibition by a second substrate molecule.

Structure of Rhizobium sp. 4-9 histamine dehydrogenase and analysis of the electron transfer pathway to an abiological electron acceptor

Histamine dehydrogenase from the gram-negative bacterium Rhizobium sp. 4-9 (HaDHR) is a member of a small family of dehydrogenases containing a covalently attached FMN, and the only member so far identified to date that does not exhibit substrate inhibition. In this study, we present the 2.1 Å resolution crystal structure of HaDHR. This new structure allowed for the identification of the internal electron transfer pathway to abiological ferrocene-based mediators. Alanine 437 was identified as the exit point of electrons from the Fe4S4 cluster. The enzyme was modified with a Ser436Cys mutation to facilitate covalent attachment of a ferrocene moiety. When modified with Fc-maleimide, this new construct demonstrated direct electron transfer from the enzyme to a gold electrode in a histamine concentration-dependent manner without the need for any additional electron mediators.

Pine has two glutamine synthetase paralogs, GS1b.1 and GS1b.2, exhibiting distinct biochemical properties.

The enzyme glutamine synthetase (EC 6.3.1.2) is mainly responsible for the incorporation of inorganic nitrogen into organic molecules in plants. In the present work, a pine (Pinus pinaster) GS1 (PpGS1b.2) gene was identified, showing a high sequence identity with the GS1b.1 gene previously characterized in conifers. Phylogenetic analysis revealed that the presence of PpGS1b.2 is restricted to the genera Pinus and Picea and is not found in other conifers. Gene expression data suggest a putative role of PpGS1b.2 in plant development, similar to other GS1b genes from angiosperms, suggesting evolutionary convergence. The characterization of GS1b.1 and GS1b.2 at the structural, physicochemical, and kinetic levels has shown differences even though they have high sequence homology. GS1b.2 had a lower optimum pH (6 vs. 6.5) and was less thermally stable than GS1b.1. GS1b.2 exhibited positive cooperativity for glutamate and substrate inhibition for ammonium. However, GS1b.1 exhibited substrate inhibition behavior for glutamate and ATP. Alterations in the kinetic characteristics produced by site-directed mutagenesis carried out in this work strongly suggest an implication of amino acids at positions 264 and 267 in the active center of pine GS1b.1 and GS1b.2 being involved in affinity toward ammonium. Therefore, the amino acid differences between GS1b.1 and GS1b.2 would support the functioning of both enzymes to meet distinct plant needs.

Assay development and inhibition of the Mt-DprE2 essential reductase from Mycobacterium tuberculosis.

DprE2 is an essential enzyme in the synthesis of decaprenylphosphoryl-β-d-arabinofuranose (DPA) and subsequently arabinogalactan, and is a significant new drug target for M. tuberculosis. Two compounds from the GSK-177 box set, GSK301A and GSK032A, were identified through Mt-DprE2-target overexpression studies. The Mt-DprE1-DprE2 complex was co-purified and a new in vitro DprE2 assay developed, based on the oxidation of the reduced nicotinamide adenine dinucleotide cofactor of DprE2 (NADH/NADPH). The Mt-DprE1-DprE2 complex showed interesting kinetics in both the DprE1 resazurin-based assay, where Mt-DprE2 was found to enhance Mt-DprE1 activity and reduce substrate inhibition; and also in the DprE2 assay, which similarly exhibited substrate inhibition and a difference in kinetics of the two potential cofactors, NADH and NADPH. Although, no inhibition was observed in the DprE2 assay by the two GSK set compounds, spontaneous mutant generation indicated a possible explanation in the form of a pro-drug activation pathway, involving fgd1 and fbiC.

Subfunctionalization of a monolignol to a phytoalexin glucosyltransferase is accompanied by substrate inhibition

Uridine diphosphate-dependent glycosyltransferases (UGTs) mediate the glycosylation of plant metabolites, thereby altering their physicochemical properties and bioactivities. Plants possess numerous UGT genes, with the encoded enzymes often glycosylating multiple substrates and some exhibiting substrate inhibition kinetics, but the biological function and molecular basis of these phenomena are not fully understood. The promiscuous monolignol/phytoalexin glycosyltransferase NbUGT72AY1 exhibits substrate inhibition (Ki) at 4 μM scopoletin, whereas the highly homologous monolignol StUGT72AY2 is inhibited at 190 μM. We therefore used hydrogen/deuterium exchange mass spectrometry and structure-based mutational analyses of both proteins and introduced NbUGT72AY1 residues into StUGT72AY2 and vice versa to study promiscuity and substrate inhibition of UGTs. A single F87I and chimeric mutant of NbUGT72AY1 showed significantly reduced scopoletin substrate inhibition, whereas its monolignol glycosylation activity was almost unaffected. Reverse mutations in StUGT72AY2 resulted in increased scopoletin glycosylation, leading to enhanced promiscuity, which was accompanied by substrate inhibition. Studies of 3D structures identified open and closed UGT conformers, allowing visualization of the dynamics of conformational changes that occur during catalysis. Previously postulated substrate access tunnels likely serve as drainage channels. The results suggest a two-site model in which the second substrate molecule binds near the catalytic site and blocks product release. Mutational studies showed that minor changes in amino acid sequence can enhance the promiscuity of the enzyme and add new capabilities such as substrate inhibition without affecting existing functions. The proposed subfunctionalization mechanism of expanded promiscuity may play a role in enzyme evolution and highlights the importance of promiscuous enzymes in providing new functions.

Kinetic modelling of high concentration 4-Nitrophenol biodegradation by an isolated bacterial consortium and post-treatment ecotoxicity analysis

The present study documents the kinetics of aerobic batch growth and biodegradation of 4-Nitrophenol (4-NP) by an isolated bacterial consortium capable of utilizing 700 mg l−1 of 4-NP as the sole source of carbon and energy, the highest reported concentration to be biodegraded till date. The results of the batch biodegradation studies suggested that the consortium exhibited substrate inhibition kinetics and the Haldane model fitted the experimental data well. The estimated biokinetic parameters applied to biomass growth were maximum specific growth rate, μmax = 0.1532 h−1, half saturation coefficient, Ks = 4.717 mg l−1, and inhibition coefficient, Ki = 77.34 mg l−1. The specific degradation rate, q, was fitted to the model, and the kinetic parameters obtained were: qmax = 0.1700 g g−1 h−1, K's = 5.871 mg l−1, and K'i = 171.9 mg l−1. The yield coefficient (YX/S) varied in the range of 0.93–0.23 g g−1 for 12–700 mg l−1 4-NP concentration. The maintenance coefficient (m) and true yield (YG) were calculated to be 0.04491 h−1 and 0.9496 g g−1, respectively, based on the Pirt's equation. Furthermore, cytotoxicity and phytotoxicity assessment involving the animal cell line HEK293 and different seed variants, respectively, confirmed the detoxification of the biodegraded product, emphasizing the biocatalytic efficiency of the consortium in the bioremediation of 4-NP contaminated wastewater and suitability of its environmental discharge.

Contributions of UDP-Glucuronosyltransferases to Human Hepatic and Intestinal Metabolism of Ticagrelor and Inhibition of UGTs and Cytochrome P450 Enzymes by Ticagrelor and its Glucuronidated Metabolite.

Ticagrelor is the first reversibly binding, direct-acting, oral P2Y12 receptor inhibitor. The contribution of UDP-glucuronosyltransferases (UGTs) enzymes to the metabolism of ticagrelor to its glucuronide conjugation, ticagrelor-O-glucuronide, in human liver microsomes (HLM) and human intestinal microsomes (HIM), was well characterized in the current study. The inhibition potential of human major UGTs by ticagrelor and ticagrelor-O-glucuronide was explored. The inhibitory effects of ticagrelor-O-glucuronide on cytochrome P450s (CYPs) enzymes were investigated as well. Ticagrelor glucuronidation exhibits substrate inhibition kinetics in both HLM and HIM with apparent Km values of 5.65 and 2.52 μM, Vmax values of 8.03 and 0.90 pmol min−1·mg protein−1, Ksi values of 1,343.0 and 292.9 respectively. The in vitro intrinsic clearances (V max/K m) for ticagrelor glucuronidation by HLM and HIM were 1.42 and 0.36 μl min−1·mg protein−1, respectively. Study with recombinant human UGTs suggested that multiple UGT isoforms including UGT1A9, UGT1A7, UGT1A3, UGT1A4, UGT1A1, UGT2B7 and UGT1A8 are involved in the conversion of ticagrelor to ticagrelor-O-glucuronide with UGT1A9 showing highest catalytic activity. The results were further supported by the inhibition studies on ticagrelor glucuronidation with typical UGT inhibitors in pooled HLM and HIM. Little or no inhibition of UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9 and UGT2B7 by ticagrelor and ticagrelor-O-glucuronide was noted. Ticagrelor-O-glucuronide also exhibited limited inhibitory effects toward CYP2C8, CYP2D6 and CYP3A4. In contrast, ticagrelor-O-glucuronide weakly inhibited CYP2B6, CYP2C9 and CYP2C19 activity with apparent IC50 values of 45.0, 20.0 and 18.8 μM, respectively. The potential of ticagrelor-O-glucuronide to cause drug-drug interactions warrant further study.

Thioredoxin regulates human mercaptopyruvate sulfurtransferase at physiologically-relevant concentrations

3-Mercaptopyruvate sulfur transferase (MPST) catalyzes the desulfuration of 3-mercaptopyruvate (3-MP) and transfers sulfane sulfur from an enzyme-bound persulfide intermediate to thiophilic acceptors such as thioredoxin and cysteine. Hydrogen sulfide (H2S), a signaling molecule implicated in many physiological processes, can be released from the persulfide product of the MPST reaction. Two splice variants of MPST, differing by 20 amino acids at the N terminus, give rise to the cytosolic MPST1 and mitochondrial MPST2 isoforms. Here, we characterized the poorly-studied MPST1 variant and demonstrated that substitutions in its Ser-His-Asp triad, proposed to serve a general acid-base role, minimally affect catalytic activity. We estimated the 3-MP concentration in murine liver, kidney, and brain tissues, finding that it ranges from 0.4 μmol·kg-1 in brain to 1.4 μmol·kg-1 in kidney. We also show that N-acetylcysteine, a widely-used antioxidant, is a poor substrate for MPST and is unlikely to function as a thiophilic acceptor. Thioredoxin exhibits substrate inhibition, increasing the KM for 3-MP ∼15-fold compared with other sulfur acceptors. Kinetic simulations at physiologically-relevant substrate concentrations predicted that the proportion of sulfur transfer to thioredoxin increases ∼3.5-fold as its concentration decreases from 10 to 1 μm, whereas the total MPST reaction rate increases ∼7-fold. The simulations also predicted that cysteine is a quantitatively-significant sulfane sulfur acceptor, revealing MPST's potential to generate low-molecular-weight persulfides. We conclude that the MPST1 and MPST2 isoforms are kinetically indistinguishable and that thioredoxin modulates the MPST-catalyzed reaction in a physiologically-relevant concentration range.

Simple and Efficient Production of Highly Soluble Daidzin Glycosides by Amylosucrase from Deinococcus geothermalis.

Transglycosylation of amylosucrase from Deinococcus geothermalis (DGAS) was performed using daidzin (daidzein-7-O-glucoside). Unlike cyclodextrin glucanotransferase, DGAS led to the production of new daidzin glucosides with high conversion yields (89%). Structures of these daidzin glucosides (i.e., DA2 and DA3) were daidzein-7-O-α-d-glucopyranosyl-(4 → 1)-O-β-d-glucopyranoside (daidzin-4″-O-α-d-glucopyranoside) and daidzein-4'-O-α-d-glucopyranosyl-7-O-α-d-glucopyranosyl-(1 → 4)-O-β-d-glucopyranoside (daidzin-4',4″-O-α-d-diglucopyranoside), respectively. DA2 and DA3 showed increased solubility of 15.4 mM (127-fold) and 203.3 mM (1686-fold) compared with daidzin, respectively. Kinetic studies revealed Vmax of 1.0 μM/min and K'm of 175 μM for DA3 production based on nonlinear regression. DGAS exhibited substrate inhibition behavior at high sucrose concentrations (700-1500 mM). Taken together, these findings indicate that DGAS can attach a glucose unit to a free C4'-OH via an α-linkage and then produce highly water-soluble isoflavone glycosides with a simple donor, moderate reaction conditions, less waste production, and high yield compared with that observed using the existing processes and enzymes.

Dissecting the Antenna in Human Glutamate Dehydrogenase: Understanding Its Role in Subunit Communication and Allosteric Regulation.

Glutamate dehydrogenase (GDH) is a homohexameric enzyme that catalyzes the reversible oxidative deamination of l-glutamate. While GDH is found in all living organisms, only that from animals is highly allosterically regulated by a wide array of metabolites. Because only animal GDH has a 50-residue antenna domain, we hypothesized that it was critical for allostery. To this end, we previously replaced the antenna with the loop found in bacteria, and the resulting chimera was no longer regulated by purine nucleotides. Hence, it seemed logical that the purpose of the antenna is to exert the subunit communication necessary for heterotrophic allosteric regulation. Here, we revisit the antenna deletion studies by retaining 10 more of the human GDH (hGDH) residues without adding the bacterial loop. Unexpectedly, the results were profoundly different than before. The basal activity of the mutant is only ∼13% of that of the wild type but ∼100 times more sensitive to all allosteric activators. In contrast, the mutant is still affected by all of the tested inhibitors to approximately the same degree. The resulting antenna-less mutant retained its negative cooperativity with respect to the coenzyme, again suggesting that intersubunit communication is intact. Finally, the mutant still exhibits substrate inhibition, albeit there are differences in the details. We present a model in which the majority of the antenna is not directly involved in allosteric regulation per se but rather may be responsible for improving enzymatic efficiency by acting as a conduit for substrate binding energy between subunits.

Kinetics of Cyanide Degradation by Azotobacter vinelandii

Cyanide is extremely toxic to living organisms. It is utilized in industries for gold and silver extraction, pharmaceuticals, plastic processing, electroplating, and agricultural chemistry. Cyanide can persevere for a long time in the soil and its bio-degradation is the best economical practice. A formerly isolated cyanide-degrading bacterium Azotobacter vinelandii exhibited substrate inhibition to degradation rate. Significant degradation inhibition constants were achieved reliably by means of non-linear regression modeling of the degradation rate profile utilizing models for substrate inhibition like Haldane, Teissier-Edward, Monod, Yano and Koga, Luong, Edward (Webb) Luong and Aiba models. Aiba model was selected as the top model established on statistical assessments like root mean square error, adjusted coefficient of determination, bias factor and accuracy factor. The calculated values for the Aiba constants qmax (the maximum specific substrate degradation rate (h−1), Ks (concentration of substrate at the half maximal degradation rate (mM) and Ki (inhibition constant (mM)) were 0.060 (95% CI, 0.024 to 0.096), 0.302 (95% CI, 0.381 to 0.222) and 0.953 (95% CI, 0.568 to 1.338), respectively. The novel constants gotten from the modeling application can be valuable for advanced secondary modeling implicating the influence of media conditions and some other dynamics on cyanide biodegradation by this bacterium.

Biosynthesis of the RiPP trojan horse nucleotide antibiotic microcin C is directed by the N-formyl of the peptide precursor.

Microcin C7 (McC) is a peptide antibiotic modified by a linkage of the terminal isoAsn amide to AMP via a phosphoramidate bond. Post-translational modification on this ribosomally produced heptapeptide precursor is carried out by MccB, which consumes two equivalents of ATP to generate the N-P linkage. We demonstrate that MccB only efficiently processes the precursor heptapeptide that retains the N-formylated initiator Met (fMet). Binding studies and kinetic measurements evidence the role of the N-formyl moiety. Structural data show that the N-formyl peptide binding results in an ordering of residues in the MccB "crossover loop", which dictates specificity in homologous ubiquitin activating enzymes. The N-formyl peptide exhibits substrate inhibition, and cannot be displaced from MccB by the desformyl counterpart. Such substrate inhibition may be a strategy to avert unwanted McC buildup and avert toxicity in the cytoplasm of producing organisms.

UGT-mediated metabolism plays a dominant role in the pharmacokinetic behavior and the disposition of morusin in vivo and in vitro

Morusin is a prenylated flavone isolated from mulberry, the branch and root bark of various Morus species, which possesses diverse pharmacological activities. However, it lacks extensive studies about its absorption and disposition. This study investigated the pharmacokinetic behavior of morusin in rat, and its first-pass metabolism in situ. The metabolic pathway of morusin was further investigated by 12 human recombinant UDP-glucuronosyltransferases (UGTs), 9 CYP450s, as well as liver and intestinal microsomes. Four mono-glucuronide metabolites (M-5-G, M-4′-G, M-2′-G, and MII-2) were identified in rat intestine and bile by LC–MS/MS, while three of them were also detected in plasma (M-5-G, M-4′-G, and MII-2). M-4′-G was the principal conjugate. However, few CYP450 metabolites were found in rat intestine and bile. Only a small amount of MI-1 could be detected in rat plasma. UGT1A1, 1A3, 1A7, and 2B7 were the major contributors to morusin glucuronidation. Morusin exhibited substrate inhibition kinetic characteristics in all UGTs. Clearance rates of M-4′-G in HLM, RLM, UGT1A1, UGT1A3, and UGT2B7 were 137.02, 127.55, 32.54, 41.18, and 35.07 ml/min/mg, respectively. Besides, CYP3A5, 3A4, and 2C19 primarily contributed to the oxidative metabolism of morusin. The pharmacokinetic curves of morusin and its conjugates presented double peaks, showing that an enterohepatic recycling may exist. In conclusion, glucuronidation was confirmed to be the crucial metabolic pathway for morusin in vivo, and M-4′-G was the main metabolite.

Expression and activities of sulfotransferase in rat brain

Sulfotransferase (SULT) has been found in the brain; however, the details of its function remain unclear. The present study aimed to elucidate the regional differences in the expression of SULT1 and SULT2 mRNA and SULT activities in the eight functional regions of the rat brain (cerebellum, cortex, hippocampus, medulla oblongata, midbrain, olfactory bulb, striatum, and thalamus).All SULT1 isoforms were detected in the medulla oblongata and thalamus. SULT2A1 mRNA was not observed in any of the eight regions, whereas SULT2B1a and SULT2B1b were found in all regions. The SULT2B1b mRNA expression level in the medulla oblongata was 1.7-fold higher than that in the liver. The sulfonation of p-nitrophenol and pregnenolone was detected in all regions. The kinetics of p-nitrophenol sulfonation in the cerebellum fitted to the substrate inhibition model (Km = 37.6 nM, Vmax = 2.72 pmol/min/mg, Vinh = 1.60 pmol/min/mg, and Ki = 0.87 μM). The pregnenolone sulfonation also exhibited substrate inhibition kinetics (Km = 0.99 μM, Vmax = 1.53 pmol/min/mg, and Ki = 54.67 μM).We clarified that SULT1 and SULT2 were expressed and had metabolizing capacities in the rat brain, suggesting that brain SULTs may be involved in metabolism of endogenous compounds and drugs.

Kinetics of growth and lipids accumulation in Chlorella vulgaris during batch heterotrophic cultivation: Effect of different nutrient limitation strategies

The present study aimed at: (1) determining the effect of sulfur addition on biomass growth and (2) assessing the effect of sulfur, phosphorus and nitrogen limitation on lipid accumulation by C. vulgaris SAG 211-11b. The sulfur cellular content was more than two-fold higher under nitrogen and phosphorus limitation (0.52% and 0.54%ww−1, respectively) compared to sulfur requirements (0.20%ww−1) under sulfur limiting conditions. The nitrogen needs are significantly lower (2.81–3.35%ww−1) when compared to other microalgae and become 23% lower under nitrogen or phosphorus limitation. The microalga exhibited substrate inhibition above 30gL−1 initial glucose concentration. Sulfur limitation had the most significant effect on lipid accumulation, resulting in maximum total lipid content of 53.43±3.93%ggDW−1. In addition to enhancing lipid productivity, adopting the optimal nutrient limitation strategy can result in cost savings by avoiding unnecessary nutrient additions and eliminate the environmental burden due to wasted resources.

Identification and biochemical characterization of the fructokinase gene family in Arabidopsis thaliana

BackgroundFructose is an abundant sugar in plants as it is a breakdown product of both major sucrose-cleaving enzymes. To enter metabolism, fructose is phosphorylated by a fructokinase (FRK). Known FRKs are members of a diverse family of carbohydrate/purine kinases known as the phosphofructokinase B (pfkB) family. The complete complement of active fructokinases has not been reported for any plant species.ResultsProtein sequence analysis of the 22 Arabidopsis thaliana pfkB members identified eight highly related predicted proteins, including one with previously demonstrated FRK activity. For one, At1g50390, the predicted open reading frame is half the size of active FRKs, and only incompletely spliced RNAs were identified, which led to a premature stop codon, both indicating that this gene does not produce active FRK. The remaining seven proteins were expressed in E. coli and phosphorylated fructose specifically in vitro leading us to propose a unifying nomenclature (FRK1–7). Substrate inhibition was observed for fructose in all FRKs except FRK1. Fructose binding was on the same order of magnitude for FRK1–6, between 260 and 480 μM. FRK7 was an outlier with a fructose Km of 12 μM. ATP binding was similar for all FRKs and ranged between 52 and 280 μM. YFP-tagged AtFRKs were cytosolic, except plastidic FRK3. T-DNA alleles with non-detectable wild-type RNAs in five of the seven active FRK genes produced no overt phenotype. We extended our sequence comparisons to include putative FRKs encoded in other plant sequenced genomes. We observed that different subgroups expanded subsequent to speciation.ConclusionsArabidopsis thaliana as well as all other plant species analyzed contain multiple copies of genes encoding FRK activity. Sequence comparisons among multiple species identified a minimal set of three distinct FRKs present on all species investigated including a plastid-localized form. The selective expansion of specific isozymes results in differences in FRK gene number among species. AtFRKs exhibit substrate inhibition, typical of their mammalian counterparts with the single AtFRK1 lacking this property, suggesting it may have a distinct in vivo role. Results presented here provide a starting point for the engineering of specific FRKs to affect biomass production.

Functional characterization of wild-type and 24 CYP2D6 allelic variants on gefitinib metabolism in vitro

BackgroundCytochrome P450 2D6 (CYP2D6), a member of the CYP450 enzyme super family, is a polymorphic enzyme that metabolizes ~25% of therapeutic drugs. CYP2D6 exhibits significant genetic polymorphisms which might cause adverse effects and therapeutic failures of some drugs.ObjectiveThe purpose of this study was to evaluate the catalytic activities of 22 novel CYP2D6 alleles (CYP2D6*87, *88, *89, *90, *91, *92, *93, *94, *95, *96, *97, *98, R25Q, F164L, E215K, F219S, V327M, D336N, V342M, R344Q, R440C, R497C) on the metabolism of gefitinib in vitro.Methods and resultsCYP2D6 variants were incubated with 1–100 μM gefitinib for 60 min at 37°C and the reaction was terminated by cooling to −80°C immediately. Gefitinib and its metabolite O-desmethyl gefitinib were analyzed by an ultra-performance liquid chromatography-tandem mass spectrometry system. Compared to CYP2D6.1, most CYP2D6 variants exhibited significantly decreased relative clearance values (from 3.11% to 79.35%), whereas CYP2D6.92 and CYP2D6.96 displayed no detectable enzyme activity. Only CYP2D6.94 exhibited a markedly increased intrinsic clearance value, and eight variants (CYP2D6.88, CYP2D6.89, CYP2D6.91, CYP2D6.97, V342M, R344Q, F219S, and F164L) showed no significant difference. In addition, 23 CYP2D6 allelic isoforms exhibited substrate inhibition trend toward gefitinib.ConclusionAs the first study of all the aforementioned alleles for gefitinib metabolism, these comprehensive data may help in the clinical assessment of the metabolism of gefitinib, and may also offer a reference for personalized treatment with gefitinib in clinical settings.

Either fadD1 or fadD2, Which Encode acyl-CoA Synthetase, Is Essential for the Survival of Haemophilus parasuis SC096.

In Haemophilus parasuis, the genes HAPS_0217 and HAPS_1695 are predicted to encode long-chain fatty acid-CoA ligases (FACSs). These proteins contain ATP/AMP signature motifs and FACS conserved motifs that are homologous to those in Escherichia coli FadD (EcFadD). In this study, we demonstrate that HAPS_0217 and HAPS_1695 can functionally replace EcFadD in the E. coli fadD mutant JW1794, and were thus designated fadD1 and fadD2, respectively. An evaluation of kinetic parameters indicated that FadD1 and FadD2 have a substrate preference for long-chain fatty acids. Moreover, FadD2 exhibited substrate inhibition in the presence of high concentrations of oleic acid. Single mutants of each of the fadD genes were easily constructed, whereas double mutants were not. These results were further confirmed using genomic site-directed mutagenesis, which supported the idea that H. parasuis requires either fadD1 or fadD2 for survival. The fadD1 mutant exhibited slower growth than the wild-type strain SC096, and its complementation resulted in a restored phenotype. The wild-type strain did not grow on chemically defined medium without the addition of oleic acid, indicating that lipids are a vital nutrient for this bacterium. Additionally, strains with a disrupted fadD1 gene also exhibited increased sensitivity to quinolone antibiotics, including levofloxacin, enrofloxacin, ciprofloxacin and nalidixic acid.

Nedd4-2 is a Functional Oligomer Exhibiting Cooperative Allosteric Kinetics

The Hect ligase Standard Model posits binding of the E2∼ubiquitin co-substrate to a single site within a monomeric Hect domain followed by transthiolation to form a Hect∼ubiquitin thioester intermediate. Transfer of the Hect-bound ubiquitin to lysyl residues on target proteins is then catalyzed by the ligase, processesively assembling polyubiquitin chains by distal addition. Recently, we demonstrated that the Hect ligase Nedd4-2 catalyzes polyubiquitin chain assembly by cooperative allosteric kinetics ([S]1/2=93±20 nM; kcat=0.067±0.008 s−1; nH=1.9±0.6) and exhibits substrate inhibition above 0.5 µM Ubc5B∼125I-ubiquitin, requiring the enzyme to function as an oligomer with two ordered E2∼ubiquitin binding sites of different affinities. These data support an alternative model for Hect ligase conjugation and are in agreement with our previous observations for E6AP. The current studies employ kinetic and biophysical methods to further explore the mechanism of Nedd4-2. Gel filtration chromatography and dynamic light scattering are consistent with trimerization. Truncation of the N-terminus yielding GST-Nedd4-2HECT (residues 597-955) reveals cooperative kinetics for polyubiquitin chain assembly (nH=2.6±1) with a ca. 104-fold reduction in kcat (1.9±0.2 x 10−6 s−1), demonstrating allostery intrinsic to the Hect domain and N-terminal contributions supporting oligomerization. Processing of GST yields monomeric Nedd4-2HECT resulting in monoubiquitination with hyperbolic kinetics (KM=39±7 nM; kcat=6.0±0.2 x 10−6 s−1) and loss of polyubiquitin chain assembly and substrate inhibition. Addition of Nedd4-2ΔHECT (residues 1-596) to wild type Nedd4-2 quantitatively inhibits activity; however, addition of the dominant negative Nedd4-2C922A mutant inhibits Nedd4-2 to 30% limiting activity, requiring the two sites to function in trans. These data support a model requiring trimerization and intersubunit interactions for polyubiquitin chain assembly consistent with our model of Proximal Indexation. [Supported by GM34009 to A.L.H.]