Deamination Reaction Research Articles

A novel in-situ chemical oxidation channel – Selective pH-dependence of refractory β-lactam antibiotics in the synergistic mechanism of persulfate and g-C3N4 under visible light

The degradation characteristics of four typical structures of β-lactam antibiotics were studied during the synergistic oxidation mechanism of persulfate (PS) and mesoporous g-C3N4 (MCN) under visible light irradiation including amoxicillin (AMX), cefotaxime (CFX), meropenem (MER), and sulbactam (SUL). Notably, the degradation of SUL in the MCN/PS system exhibits a pH-dependence different from the other three antibiotics. In this work, a novel oxidation channel for such persistent organic pollutant was revealed. Initially, SUL was bound specifically with PS via the addition reaction caused by the electron transfer between the oxygen atoms of the sulfoxide group of SUL and the HSO3− group generated by the protonation of PS. The reaction was carried out under acidic conditions and generated the SUL-O-SO2H byproducts. Successively, the adduct was further oxidized by the free radicals and singlet oxygen until the intermediate products were completely mineralized. The environmental factors of the reaction, such as the presence of humic acid and the cation-anion interactions, were also considered. In this work, the degradation pathways of SUL, the hydrolysis of the β-lactam ring, the decarboxylation, and the deamination reaction based on the combination of DFT calculations and the HPLC-QTOF-MS/MS analysis are elucidated. The toxicity of the byproducts was also calculated via the USEPA TEST. The findings improve the understanding of the reaction mechanism of the persulfate and the photocatalytic systems and provide new ideas for the oxidative degradation of refractory organic contaminants.

Removal of crystal violet by ultraviolet/persulfate: Effects, kinetics and degradation pathways

Abstract The degradation of crystal violet (CV) by the ultraviolet/persulfate (UV/PS) process was investigated in this study. The degradation processes of CV by PS and UV/PS could be appropriately described by second-order degradation kinetics, with kinetics rate constants of 3.57 × 10−3 and 9.58 × 10−3 g⋅mg−1min−1, respectively (UV lamp at 0.346 mW/cm2, CV 0 = 10 mg/L, PS = 0.5 mmol/L, pH = 7.0, and temperature = 20 °C). An increases in PS dosage and temperature can both promote the degradation of CV. However, an excessive PS dosage ( ≥ 1 mmol/L), too high a temperature ( ≥ 25 °C) and too low an initial CV concentration may not be desirable options with respect to the economic cost. In addition, better degradation efficiencies were obtained both under neutral conditions (pH = 7) and the presence of an appropriate amount of metal ions (0.1 mmol/L), such as Cu 2 + , Zn 2 + , and Fe2＋. Economical cost comparison revealed that UV/PS has a better economy as compared to UV/H2O2 and UV/TiO2. Free radical quenching experiments revealed that the contributions of ⋅OH and SO⋅ 4 − to the CV removal by UV/PS process were 19.7% and 26%, respectively (UV lamp at 0.346 mW/cm2, CV 0 = 10 mg/L, PS = 0.5 mmol/L, pH = 7.0, and temperature = 20 °C). The CV degradation pathways of UV/PS were proposed and can be summarized as follows: demethylation, decarboxylation, deamination and ring-opening reaction following the oxidative cleavage of the double bond of CV, after which small molecules such as H2O, CO 3 2 − , NO 3 − were eventually generated.

Examining the Structure of Activation‐Induced Cytidine Deaminase (AID)

In order to respond to any foreign antigen that might present itself, B cells must produce antibodies of great variety. Activation‐Induced Cytidine Deaminase (AID) is a protein that plays a critical role in the diversification of antibodies.1 Discovered by T. Honjo et al. in 2000, AID is a protein consisting of 198 amino acids that creates a U:G mismatch by deaminating cytosine to uracil in transcribed Ig DNA2. AID helps in the process of binding site differentiation, known as somatic hypermutation (SHM), and class‐switching, known as CSR (class switch DNA recombination).3 The Governor’s Academy MSOE Center for BioMolecular Modeling SMART team used 3‐D modeling and printing technology to better understand how several aspects of the structure of AID play a critical role in accomplishing the functions of SHM and CSR. Our model highlights Histidine 56, Cysteine 87 and Cysteine 90, which bind zinc ions forming a zinc finger, and the hotspot recognition loop, all of which are involved in DNA binding.4 The catalytic domain, which is similar to the other members of the apolipoprotein B RNA‐editing cytidine deaminase enzymes (APOBEC) is also shown.5 Unfortunately, two of the residues that are most critical for AID functioning could not be shown on our model. Serine 38, which is an important phosphorylation site, was cut out during the isolation and crystallization process and thus is not included in the protein data bank files. Glutamic Acid 58 is the residue most critical for the deamination reaction and was mutated to an Alanine in order to prevent the reaction from occurring so that AID could be isolated with DNA bound.6 Lastly, we highlighted residues R112 and R24. Mutation of either of these Arginines disrupts the configuration of AID, which undermines the ability of AID to undergo SHM and CSR, causing an immunodeficiency disorder called Hyper‐IgM syndrome type 2 (HIGM‐2)

What is the Functional Role of Organelle Zinc finger 1 in the Plant C‐to‐U RNA Editing Complex?

Higher plants utilize protein complexes to precisely edit hundreds of RNA transcripts in organelles. The mechanistic model of RNA editing involves a C‐to‐U deamination reaction facilitated through the interaction of the RNA with a protein complex. Complex models have been proposed, but the minimal “editosome” capable of catalyzing C‐to‐U changes and the functions of many of the protein components have not been revealed. This research focuses on describing the functional role of one critical protein component belonging to the RAN binding protein 2 type zinc fingers protein family, Organelle Zinc finger 1 (OZ1). Zinc finger proteins have been associated with RNA binding; though RNA binding and site recognition in editosomes is known to occur via PPR proteins, OZ1 may be interacting with a scaffold RNA sequence. Recombinant OZ1 from Arabidopsis thaliana was used to analyze zinc coordination and RNA binding of this zinc finger protein. Spectrophotometric analysis of OZ1 after immobilized metal affinity chromatography (IMAC) suggests nucleic acid association. ICP‐MS and ESI‐MS analysis of a truncated variant of OZ1, only the sequence containing its two zinc finger domains (OZ1zf), suggests that OZ1 is coordinating only one zinc ion instead of the proposed two. Computational analysis of OZ1 dependent sites in Brachypodium distachyon returned a potential motif conserved among OZ1‐dependent editing sites. Electrophoretic mobility shift assays (EMSAs) are used to test RNA binding and the motif. Understanding this protein will bring us a step closer to manipulating this precise editing machine for use in biotechnology and may be used for purposes of crop improvement.Support or Funding InformationFunding for this work was provided by NIH SCORE SC2 ID: SC2 GM122718 awarded to Dr. Michael L. Hayes and the NIH MBRS‐RISE R25 GM 061331‐17 grant in support of Alfredo Jimenez‐Salinas.

Nitrogen Metabolism in Cancer and Immunity.

As one of the fundamental requirements for cell growth and proliferation, nitrogen acquisition and utilization must be tightly regulated. Nitrogen can be generated from amino acids (AAs) and utilized for biosynthetic processes through transamination and deamination reactions. Importantly, limitations of nitrogen availability in cells can disrupt the synthesis of proteins, nucleic acids, and other important nitrogen-containing compounds. Rewiring cellular metabolism to support anabolic processes is a feature common to both cancer and proliferating immune cells. In this review, we discuss how nitrogen is utilized in biosynthetic pathways and highlight different metabolic and oncogenic programs that alter the flow of nitrogen to sustain biomass production and growth, an important emerging feature of cancer and immune cell proliferation.

Hydrolytic deamination mechanisms of guanosine monophosphate: A computational study

The computational importance of guanosine monophosphate (GMP) comes from its importance in the synthesis of nucleic acids. Deamination reaction mechanisms, kinetics, and thermodynamics of GMP with 3H2O and 2H2O/OH− to produce xanthosine monophosphate (XMP) have been investigated. The proposed reactions were optimized in the gas-phase and in an aqueous medium. Two key steps were found for the proposed pathways: a tetrahedral intermediate formation and a single water-mediated six-membered ring transition state, yielding the desired product. The deamination of GMP with 2H2O/OH− yield deprotonated XMP− with only one pathway, which is highly exothermic, and the activation energy is significantly lower (33 kJ mol−1 at M11/6-31G(d)) compared to the other pathways. Our results suggest that pathway C is the most kinetically and thermodynamically favorable mechanism for the deamination of GMP with 3H2O with the lowest energy barrier of 123 kJ mol−1, due to multiple functions of water that enhance the reaction profile.

Catalytic co-pyrolysis of seaweeds and cellulose using mixed ZSM-5 and MCM-41 for enhanced crude bio-oil production

Catalytic co-pyrolysis of seaweed Enteromorpha clathrata (EN) and cellulose (CEL) with catalysts ZSM-5 and MCM-41 was investigated by TG, Py–GC/MS and fixed-bed experiments. The effects of temperature, catalysts, seaweed and cellulose ratio were examined on product yields distribution and bio-oil compositions by catalytic co-pyrolysis. The maximum bio-oil yield was recorded at the ratio of 1:1 (EN and CEL) with ZSM-5/MCM-41 at 500 °C on co-pyrolytic process. The interaction of radicals and faster heat transfer rate of EN/CEL induces the synergistic effects with catalysts. The advantage of mesoporous molecular sieve along with acidic microporous zeolite of ZSM-5/MCM-41 improved the cracking, dehydration, decarbonylation, decarboxylation, dealkylation, aromatization, oligomerization and deamination reactions. The overall study revealed that the amount of N-containing compounds were decreased and significantly elevated bio-oil production with increased furans and aromatics.

Identification of soybean trans-factors associated with plastid RNA editing sites.

RNA editing is a posttranscriptional process that changes nucleotide sequences, among which cytosine-to-uracil by a deamination reaction can revert non-neutral codon mutations. Pentatricopeptide repeat (PPR) proteins comprise a family of RNA-binding proteins, with members acting as editing trans-factors that recognize specific RNA cis-elements and perform the deamination reaction. PPR proteins are classified into P and PLS subfamilies. In this work, we have designed RNA biotinylated probes based in soybean plastid RNA editing sites to perform trans-factor specific protein isolation. Soybean cis-elements from these three different RNA probes show differences in respect to other species. Pulldown samples were submitted to mass spectrometry for protein identification. Among detected proteins, five corresponded to PPR proteins. More than one PPR protein, with distinct functional domains, was pulled down with each one of the RNA probes. Comparison of the soybean PPR proteins to Arabidopsis allowed identification of the closest homologous. Differential gene expression analysis demonstrated that the PPR locus Glyma.02G174500 doubled its expression under salt stress, which correlates with the increase of its potential rps14 editing. The present study represents the first identification of RNA editing trans-factors in soybean. Data also indicated that potential multiple trans-factors should interact with RNA cis-elements to perform the RNA editing.

Crystal structure of the reactive intermediate/imine deaminase A homolog from the Antarctic bacterium Psychrobacter sp. PAMC 21119

The RidA subfamily proteins catalyze the deamination reaction of enamine/imine intermediates, which are metabolites of amino acids such as threonine and serine. Numerous structural and functional studies have been conducted on RidA isolated from mesophiles and thermophiles. However, little is known about the structure of the RidA proteins isolated from psychrophiles. In the present study, we elucidated the crystal structure of RidA from the Antarctic bacterium Psychrobacter sp. PAMC 21119 (Pp-RidA) at 1.6 Å resolution to identify the structural properties contributing to cold-adaptability. Although the overall structure of Pp-RidA is similar to those of its homologues, it exhibits specific structural arrangements of a loop positioned near the active site, which is assumed to play a role in covering the active site of catalysis. In addition, the surface electrostatic potential of Pp-RidA suggested that it exhibits stronger electrostatic distribution relative to its homologues. Our results provide novel insights into the key determinants of cold-adaptability.

Hydrolytic deamination reactions of amidine and nucleobase derivatives

AbstractAmidines share the same NC─N building framework with many essential biochemical substances. In this work, we present a comparative mechanistic study on the deamination reactions of 19 amidine and nucleobase derivatives by the use of density functional theory. All the computations are performed at the B3LYP/6‐31G(d,p) level in the gas phase and with the polarizable continuum model (PCM). Mechanisms of 2‐ and 3‐step pathways including six‐ or eight‐membered ring transition states were explored. Our results show that the overall activation energies for the deamination of amidine derivatives are close to those of nucleobase derivatives of the saturated C5─C6 bond, and lower than those of nucleobase derivatives of the unsaturated C5─C6 bond, while purine derivatives have the highest activation energies among all the derivatives studied. The 3‐step mechanism gives results that are more consistent with the available experimental data than the 2‐step mechanism. Based on the results of our current and previous work, we believe that the 3‐step mechanism is the most likely mechanism for the hydrolytic deamination reactions of amidine and nucleobase derivatives.

Abstract 825: TAPS: A bisulfite-free, base-resolution and quantitative sequencing method for cytosine modifications

Abstract Aberrant DNA methylation changes are powerful prognostic and predictive biomarkers in cancer diagnosis and treatment. For decades bisulfite sequencing has been the gold standard for DNA methylation analysis. However, this method relies on harsh deamination reaction of unmodified cytosine to uracil which degrades the majority of DNA and generates sequencing libraries with low complexity. Those drawbacks limit the applications on low-input samples and lead to poor sequencing quality, low mapping rates, uneven genome coverage and increased sequencing cost. Here, we present a novel bisulfite-free and base-resolution sequencing method, TET Assisted Pyridine borane Sequencing (TAPS), for direct detection of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC) without affecting unmodified cytosine. We utilised TET enzymes to readily oxidize 5mC and 5hmC to 5-carboxylcytosine (5caC), which was then converted to dihydrouracil by pyridine borane via a previously unknown reductive decarboxylation/deamination reaction. This non-destructive method enables high-throughput sequencing with nanoscale input materials (genomic DNA and cell-free DNA) and preserve DNA up to 10 kbs long which could allow long read sequencing. We demonstrated that TAPS could directly replace bisulfite sequencing with higher mapping rates, more even coverage and lower sequencing costs, and enable more comprehensive, cheaper and faster methylome analyses. This would be a revolutionary tool for epigenetic study and facilitate both academic research and clinical diagnostics. Citation Format: Yibin Liu, Paulina Siejka, Gergana Velikova, Ying Bi, Marketa Tomkova, Benjamin Schuster-Boeckler, Chun-Xiao Song. TAPS: A bisulfite-free, base-resolution and quantitative sequencing method for cytosine modifications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 825.

Electrochemical removal of metribuzin in aqueous solution by a novel PbO2/WO3 composite anode: Characterization, influencing parameters and degradation pathways

Abstract In this work, we fabricated PbO2/WO3 composite electrode through composite electrodeposition method and employed it in electrocatalytic degradation of metribuzin. The effects of WO3 particles on surface morphology, structure and electrochemical characters of PbO2 electrodes were investigated. Different from PbO2 electrodes, the PbO2/WO3 composite electrodes owned higher oxygen evolution overpotential, longer service life and higher hydroxyl radicals generation performance. The effect of key operating parameters, for instance, current density, initial metribuzin concentration, initial pH values, and supporting electrolyte concentration, upon electrochemical removal process of metribuzin were studied. The current density and initial metribuzin concentration exerted prominent effects on electrochemical degradation of metribuzin. Kinetic analysis indicated that the electrochemical decomposition of metribuzin was a pseudo-first-order reaction. The byproducts during metribuzin degradation process were identified, several aromatic products and inorganic ions were generated along with the reaction time. Finally, the electrochemical pathways of metribuzin degradation at the PbO2/WO3 composite electrode were proposed based on the identified reaction byproducts, which were separated into three parallel sub-pathways according to the different degradation reactions by hydroxyl radicals, and quantities of hydroxylation, oxidation, demethylation, deamination, and addition reactions’ products were generated. All the byproducts were finally decomposed into inorganic H2O, CO2, NO3−, and NH4+.

Investigation on co-pyrolysis of lignocellulosic biomass and amino acids using TG-FTIR and Py-GC/MS

Co-pyrolysis of lignocellulosic biomass and microalgae could greatly improve bio-oil quality. In order to understand the interaction, nitrogen transformation mechanism, and better use lignocellulosic biomass and microalgae, co-pyrolysis of lignocellulosic biomass (bamboo waste (Ba)) and amino acids (glutamic acid (Glu) and aspartic acid (Asp)) was performed by TG-FTIR and Py-GC/MS technique. Results showed that individual pyrolysis of Glu and Asp formed 52% pyrrolidinone and 75% maleimide through intramolecular dehydration reactions, respectively, with the releasing of CO2, CO and NH3. Besides, co-pyrolysis of Ba with Glu and Asp decreased the biomass decomposition temperature largely. Furthermore, co-pyrolysis greatly increased the yield of CO2 and NH3, and promoted the formation of phenols, O-containing species (but oxygen yield decreased), while inhibited the generation of N-species (decreased about 50%) in bio-oil, through strong Maillard reaction, ketonization, deamination, and decarboxylation reactions. At last, the possible reaction pathways of co-pyrolysis of lignocellulosic biomass and amino acids were proposed. In conclusion, co-pyrolysis of lignocellulosic biomass and amino acids could significantly improve the quality of bio-oil, and effectively control the nitrogen transformation into bio-oil.

Formation of Quinone Methides by Ultrafast Photodeamination: A Spectroscopic and Computational Study.

Formation of quinone methides (QMs) by photoelimination of an ammonium salt from cresol derivatives was investigated by femtosecond transient absorption spectroscopy (fs-TA) and computationally by time-dependent density functional theory using the PCM(water)/(TD-)B3LYP/6-311++G(d,p) level of theory. The photoelimination takes place in an adiabatic ultrafast reaction on the S1 potential energy surface delivering the corresponding QMs(S1), which were detected by fs-TA. Computations predicted a high-energy cation intermediate in the pathway between the Franck-Condon state of a monoammonium salt and the corresponding QM(S1) that was not detected by fs-TA. On the other hand, elimination from a disalt in H2O takes place in one step, giving directly the QM(S1). The combined experimental and theoretical investigation fully disclosed the formation of QMs by the deamination reaction mechanism, which is important in the application of cresols or similar molecules in biological systems.

Cometabolic biotransformation and microbial-mediated abiotic transformation of sulfonamides by three ammonia oxidizers

The abilities of three phylogenetically distant ammonia oxidizers, Nitrososphaera gargensis, an ammonia-oxidizing archaeon (AOA); Nitrosomomas nitrosa Nm90, an ammonia-oxidizing bacterium (AOB); and Nitrospira inopinata, the only complete ammonia oxidizer (comammox) available as a pure culture, to biotransform seven sulfonamides (SAs) were investigated. The removals and protein-normalized biotransformation rate constants indicated that the AOA strain N. gargensis exhibited the highest SA biotransformation rates, followed by N. inopinata and N. nitrosa Nm90. The transformation products (TPs) of sulfadiazine (SDZ), sulfamethazine (SMZ) and sulfamethoxazole (SMX) and the biotransformation mechanisms were evaluated. Based on the analysis of the TP formulas and approximate structures, it was found that during biotransformation, i) the AOA strain carried out SA deamination, hydroxylation, and nitration; ii) the AOB strain mainly performed SA deamination; and iii) the comammox isolate participated only in deamination reactions. It is proposed that deamination was catalyzed by deaminases while hydroxylation and nitration were mediated by nonspecific activities of the ammonia monooxygenase (AMO). Additionally, it was demonstrated that among the three ammonia oxidizers, only AOB contributed to the formation of pterin-SA conjugates. The biotransformation of SDZ, SMZ and SMX occurred only when ammonia oxidation was active, suggesting a cometabolic transformation mechanism. Interestingly, SAs could also be transformed by hydroxylamine, an intermediate of ammonia oxidation, suggesting that in addition to enzymatic conversions, a microbially induced abiotic mechanism contributes to SA transformation during ammonia oxidation. Overall, using experiments with pure cultures, this study provides important insights into the roles played by ammonia oxidizers in SA biotransformation.

Characterization of Alanine Dehydrogenase and Its Effect on Streptomyces coelicolorA3(2) Development in Liquid Culture

Streptomyces, the most important group of industrial microorganisms, is harvested in liquid cultures for the production of two-thirds of all clinically relevant secondary metabolites. It is demonstrated here that the growth of Streptomyces coelicolor A3(2) is impacted by the deletion of the alanine dehydrogenase (ALD), an essential enzyme that plays a central role in the carbon and nitrogen metabolism. A long lag-phase growth followed by a slow exponential growth of S. coelicolor due to ALD gene deletion was observed in liquid yeast extract mineral salt culture. The slow lag-phase growth was replaced by the normal wild-type like growth by ALD complementation engineering. The ALD enzyme from S. coelicolor was also heterologously cloned and expressed in Escherichia coli for characterization. The optimum enzyme activity for the oxidative deamination reaction was found at 30°C, pH 9.5 with a catalytic efficiency, k_cat/K_M, of 2.0 ± 0.1 mM^–1 s^–1. The optimum enzyme activity for the reductive amination reaction was found at 30°C, pH 9.0 with a catalytic efficiency, k_cat/K_M, of 1.9 ± 0.1 mM^–1 s^–1.

Acid activated montmorillonite for gas-phase catalytic dehydration of monoethanolamine

The acid activated montmorillonite (Acid-Mt) was investigated for the first time as a catalyst for the gaseous-phase dehydration of monoethanolamine (MEA). A series of Acid-Mt was obtained by treating the Na-type montmorillonite (NaMt) at 104 °C in 3.2 mol L−1 HNO3 aqueous solution for 4–24 h followed by calcining at 450–1150 °C for 4 h, respectively. The Acid-Mts were characterized by X-ray diffraction, temperature programmed desorption of NH3, temperature programmed desorption of CO2, and N2 adsorption-desorption, and were evaluated as catalysts for the gaseous-phase dehydration of MEA. With increasing the activation time or calcination temperature, both the amounts of acidic and basic sites over Acid-Mt were decreased, and more pronounced extent was observed in the case of changing the calcination temperature. By correlating the catalytic results with the characterization results, the acidity of the catalyst was revealed to be the key factor in determining the MEA conversion. In contrast, a complex picture was observed in the case of the product selectivity. Generally, a higher acidity over Acid-Mt favored the intermolecular dehydration and 1,2-elimination reactions of MEA, leading to a higher selectivity of piperazine (PIP) and triethylenediamine (TEDA). When the acidity of Acid-Mt was lower, the intramolecular dehydration and deamination reactions were improved resulting in an increasing selectivity of ethyleneimine (EI). Importantly, the product distribution of the MEA dehydration was greatly regulated by the simply changing the acid activation parameters of Mt. Thus, Acid-Mt is a promising catalyst for selectively synthesizing different N-containing fine chemicals via the MEA dehydration reaction.

Onset of deaminase APOBEC3B induction in response to DNA double-strand breaks

Deamination of 5-methyl cytosine is a major cause of cancer-driver mutations in inflammation-associated cancers. The deaminase APOBEC3B is expressed in these cancers and causes mutations under replication stress; however, the mechanisms by which APOBEC3B mediates deamination and its association with genomic disorders are still unclear. Here, we show that APOBEC3B is stabilized to induce deamination reaction in response to DNA double-strand breaks (DSBs), resulting in the formation of long-lasting DSBs. Uracil, the major deamination product, is subsequently targeted by base excision repair (BER) through uracil-DNA glycosylase 2 (UNG2); hence late-onset DSBs arise as by-products of BER. The frequency of these delayed DSBs was increased by treatment of cells with a PARP inhibitor, and was suppressed following knock-down of UNG2. The late-onset DSBs were induced in an ATR-dependent manner. Those secondary DSBs were persistent, unlike DSBs directly caused by γ-ray irradiation. Overall, these results suggest that the deaminase APOBEC3B is induced in response to DSBs, leading to long-lasting DSB formation in addition to mutagenic 5me-C>T transition induction.

Metabolic repair through emergence of new pathways in Escherichia coli

Escherichia coli can derive all essential metabolites and cofactors through a highly evolved metabolic system. Damage of pathways may affect cell growth and physiology, but the strategies by which damaged metabolic pathways can be circumvented remain intriguing. Here, we use a ΔpanD (encoding for aspartate 1-decarboxylase) strain of E. coli that is unable to produce the β-alanine required for CoA biosynthesis to demonstrate that metabolic systems can overcome pathway damage by extensively rerouting metabolic pathways and modifying existing enzymes for unnatural functions. Using directed cell evolution, rewiring and repurposing of uracil metabolism allowed formation of an alternative β-alanine biosynthetic pathway. After this pathway was deleted, a second was evolved that used a gain-of-function mutation on ornithine decarboxylase (SpeC) to alter reaction and substrate specificity toward an oxidative decarboxylation-deamination reaction. After deletion of both pathways, yet another independent pathway emerged using polyamine biosynthesis, demonstrating the vast capacity of metabolic repair.

Ultra-acceleration of Photochemical Cytosine Deamination by Using a 5'-Phosphate-Substituted Oligodeoxyribonucleotide Probe Containing a 3-Cyanovinylcarbazole Nucleotide at Its 5'-End.

Genes are the blueprints for the architectures of living organisms, providing the backbone of the information required for formation of proteins. Changes in genes lead to disorders, and these disorders could be rectified by reversing the mutations that caused them. Photochemical methods currently in use for site-directed mutagenesis employ the photoactive 3-cyanovinylcarbazole (CNV K) nucleotide incorporated in the oligodeoxyribonucleotide (ODN) backbone. The major drawback of this method, the requirement for high temperature, has been addressed, and deamination has previously been achieved at 37 °C but with low efficiency. Here, efficient deamination has been accomplished under physiological conditions by using a short complementary photoactive ODN with a 5'-phosphate group in the -1 position with respect to the target cytosine. It is hypothesized that the free phosphate group affects the microenvironment around the target cytosine by activating the incoming nucleophile through hydrogen bonding with the water molecule, thus facilitating nucleophilic attack on the cytosine C-4 carbon. The degree of deamination observed in this technique is high and the effect of the phosphate group is to accelerate the deamination reaction.