Modification Of Side Chains Research Articles

Serrulatane diterpenoids with unusual side chain modifications from root bark of Eremophila longifolia

The plant genus Eremophila is endemic to Australia and widespread in arid regions. Root bark extract of Eremophila longifolia (R.Br.) F.Muell. (Scrophulariaceae) was investigated by LC-PDA-HRMS, and dereplication suggested the presence of a series of diterpenoids. Using a combination of preparative- and analytical-scale HPLC separation as well as extensive 1D and 2D NMR analysis, the structures of 12 hitherto unreported serrulatane diterpenoids, eremolongine A-L, were established. These structures included serrulatanes with unusual side chain modifications to form hitherto unseen skeletons with, e.g., cyclopentane, oxepane, and bicyclic hexahydro-1H-cyclopenta[c]furan moieties. Serrulatane diterpenoids in Eremophila have recently been shown to originate from a common biosynthetic precursor with conserved stereochemical configuration, and this was used for tentative assignment of the relative and absolute configuration of the isolated compounds. Triple high-resolution α-glucosidase/α-amylase/PTP1B inhibition profiling demonstrated that several of the eremolongines had weak inhibitory activity towards targets important for management of type 2 diabetes.

Effect of counter anions and side-chain modification of conjugated polymers for organic solar cells as an interlayer: An in-depth investigation of the diverse modification of ionic functionality

A conjugated polymer based on dimethylaminopropyl fluorene and naphthalene diimide (PFN-NDI) was synthesized to apply for organic solar cells (OSC) as the interlayer. Further, we convert the amine group to quaternary ammonium salt with various types of counter anion (CA) such as bromide and iodide, in addition to the side chain modification with hydroxyl groups. These materials generate a favorable interface dipole by the ionic functionality at the end of the side chain, while the polar hydroxyl groups increase the magnitude of interface dipole. The power conversion efficiency (PCE) was improved by incorporating polyelectrolytes as the interlayer. The PCE of the device based on PFN-NDI with acetic acid additive as the interlayer was enhanced over the device with pristine ZnO from 8.40% to 8.51%. The PCE of the device based on PFN-NDI-Br was enhanced up to 9.02%, due to the significant improvement of FF. The incorporation of extra hydroxyl groups increases the PCE up to 9.15% as a result of the synergy effect in the interface dipole moment. Furthermore, the PCE improvement was obtained by using PFN-NDI-I as the interlayer, up to 9.41%, which is mainly resulted from the Jsc enhancement. This result suggests a correlation between device performance and ionic functionality modification.

The Changes Occurring in Proteins during Processing and Storage of Fermented Meat Products and Their Regulation by Lactic Acid Bacteria.

Protein, which is the main component of meat, is degraded and oxidized during meat fermentation. During fermentation, macromolecular proteins are degraded into small peptides and free amino acids, and oxidation leads to amino acid side chain modification, molecular crosslinking polymerization, and peptide chain cleavage. At different metabolic levels, these reactions may affect the protein structure and the color, tenderness, flavor, and edible value of fermented meat products. Lactic acid bacteria are currently a research hotspot for application in the fermented meat industry. Its growth metabolism and derivative metabolites formed during the fermentation of meat products regulate protein degradation and oxidation to a certain extent and improve product quality. Therefore, this paper mainly reviews the changes occurring in proteins in fermented meat products and their effects on the quality of the products. Referring to studies on the effects of lactic acid bacteria on protein degradation and oxidation from all over the world, this review aims to provide a relevant reference for improving the quality of fermented meat products.

Production and Functional Characterization of a Novel Mannanase from Alteromonadaceae Bacterium Bs31.

Mannans are the main components of hemicellulose in nature and serve as the major storage polysaccharide in legume seeds. To mine new mannanase genes and identify their functional characteristics are an important basis for mannan biotechnological applications. In this study, a putative mannanase gene (ManBs31) from the genome of the marine bacterium Alteromonadaceae Bs31 was characterized. Amino acid sequence analysis and protein structural modeling were used to reveal the molecular features of ManBs31. The catalytic domain of ManBs31 was recombinantly produced using Escherichia coli and Pichia pastoris expression systems. The biochemical properties of the enzymes were determined by reducing sugar assay and thin-layer chromatography. Sequence analysis revealed that ManBs31 was a multidomain protein, consisting of a catalytic domain belonging to glycoside hydrolase family 5 (GH5) and two cellulose-binding domains. Recombinant ManBs31-GH5 exhibited the maximum hydrolytic performance at 70 ºC and pH 6. It showed the best hydrolysis capacity toward konjac glucomannan (specific enzyme activity up to 1070.84 U/mg) and poor hydrolysis ability toward galactomannan with high side-chain modifications (with a specific activity of 344.97 U/mg and 93.84 U/mg to locust bean gum and ivory nut mannan, respectively). The hydrolysis products of ManBs31-GH5 were mannooligosaccharides, and no monosaccharide was generated. Structural analysis suggested that ManBs31-GH5 had a noncanonical +2 subsite compared with other GH5 mannanases. ManBs31 was a novel thermophilic endo-mannanase and it provided a new alternative for the biodegradation of mannans, especially for preparation of probiotic mannooligosaccharides.

Side-chain modifications of phyllobilins may not be essential for chlorophyll degradation in Arabidopsis.

Disposing efficiently and safely chlorophyll derivatives during senescence requires a coordinated pathway that is well conserved throughout green plants. The PAO/phyllobilin pathway catalyzes the degradation of the chlorophyll during senescence and allows detoxification of the pigment and its subsequent export from the chloroplast. Although most of the chloroplastic reactions involved in chlorophyll degradation are well understood, the diversity of enzymes responsible for downstream modifications of non‐phototoxic phyllobilins remains to be explored. More than 40 phyllobilins have been described to date, but only three enzymes catalyzing side‐chain reactions have been identified in Arabidopsis thaliana , namely, TIC55, CYP89A9, and MES16. Here, by generating a triple mutant, we evaluate the extent to which these enzymes are influencing the rate and amplitude of chlorophyll degradation at the metabolite as well as its regulation at the transcriptome level. Our data show that major side‐chain modifications of phyllobilins do not influence significantly chlorophyll degradation or leaf senescence, letting the physiological relevance of their striking diversity an open question.

Computational Analysis Comparison Prediction of Anticancer Peptide (ACP)

Abstract: Anticancer peptides (ACPs) contain short peptides composed of 10–60 amino acids that can inhibit tumors cell proliferation or migration, or suppress the formation of tumors blood vessels, and are less likely to cause drug resistance. The aforementioned merits make ACPs the most promising anti-cancer candidate. ACPs may be degraded by proteases and result in cytotoxicity in many cases. To overcome these drawbacks, a plethora of research has focused on the reconstruction or modification of ACPs to improve their anti-cancer activity, while reducing their cytotoxicity. ACPs modification mainly includes main chain reconstruction and side-chain modification. After summarizing the classification and mechanism of action of ACPs, this paper focuses on recent development and progress in their reconstruction and modification. The information collected here may provide some ideas for further research on ACPs, in particular their modification. ACPs were extracted from various search engines like PubMed, Google scholar and Patent lens. Specific searches were carried out using a combination of keywords like ‘ACPs’, ‘antitumor peptides’, ‘anti-angiogenic peptides’, ‘anti-metastatic peptides’ and ‘host defence peptides. The comprehensive information related to a peptide like its source of origin, nature of the peptide, anticancer activity, N- and Cterminal modifications, conformation, etc. Additionally, CancerPPD provides information on around 249 types of cancer cell lines and 16 different assays used for testing the ACPs. In natural peptides, CancerPPD contains peptides having non-natural, chemically modified residues and D-amino acids. Besides this primary information, CancerPPD stores predicted tertiary structures as well as peptide sequences in SMILES format.

Relative reinforcing effects of dibutylone, ethylone, and N-ethylpentylone: self-administration and behavioral economics analysis in rats.

Following the emergence of methylone as one of the most popular synthetic cathinones, this group of novel psychoactive substance with names ending in "-lone," such as dibutylone, ethylone, and N-ethylpentylone, appeared on the recreational drug market. The pharmacological mechanisms of dibutylone, ethylone, and N-ethylpentylone are well understood; however, to date, the reinforcing effects of dibutylone, ethylone, and N-ethylpentylone are still unclear. This study aimed to examine the self-administration of dibutylone, ethylone, and N-ethylpentylone relative to methamphetamine (METH) and to quantify their relative reinforcing effectiveness using behavioral economic analysis. Male Sprague-Dawley rats were trained to self-administer METH (0.05mg/kg) under a fixed-ratio 1 (FR1) schedule. Following the training, dose substitution was used to generate full dose-response curves for METH and the three synthetic cathinones. According to the first doses on the descending limb of the dose-response curves, rats were trained to self-administer METH (0.05mg/kg), dibutylone (0.1mg·kg-1·infusion-1), ethylone (0.4mg·kg-1·infusion-1), or N-ethylpentylone (0.1mg·kg-1·infusion-1) under an FR1 schedule, and a behavioral economic evaluation of their reinforcing effectiveness was then performed. Dibutylone, ethylone, and N-ethylpentylone functioned as reinforcers, and the inverted U-shaped dose-response curves were obtained. The rank order of reinforcing potency in this procedure was METH > N-ethylpentylone ≈ dibutylone > ethylone. In the economic analysis, the comparisons of the essential value (EV) transformed from demand elasticity (α) indicated that the rank order of efficacy as reinforcers was METH (EV = 7.93) ≈ dibutylone (EV = 7.81) > N-ethylpentylone (EV = 5.21) ≈ ethylone (EV = 4.19). These findings demonstrated that dibutylone, ethylone, and N-ethylpentylone function as reinforcers and have addictive potential, suggesting that the modification of α-alkyl and N-alkyl side chains may affect their reinforcing efficacy.

Clickable, Oxidation-Responsive and Enzyme-Degradable Polypeptide: Synthesis, Characterization and Side Chain Modification

Clickable, Oxidation-Responsive and Enzyme-Degradable Polypeptide: Synthesis, Characterization and Side Chain Modification

Characterization of hemicellulose during xylogenesis in rare tree species Castanopsis hystrix

Hemicellulose is an important component of the plant cell wall which vary in structure and composition between plant species. The research of hemicellulose structures is primarily focused on fast-growing plants during xylogenesis, with slow-growing and rare trees receiving the least attention. Here, hemicellulose structure of the rare species Castanopsis hystrix during xylogenesis was analyzed. Acetyl methyl glucuronide xylan was the most common type of hemicellulose in C. hystrix, with a unique tetrasaccharide structure at the reducing end. Hemicellulose type, structure, molecular weight, thermal stability, biosynthesis and acetyl substitution content and pattern remained stable during the xylogenesis in C. hystrix, which could be attributed to its slow growth. The stable polymer type, low side chain modification and high acetyl substitution of hemicellulose throughout the stems are among the reasons for the hardness and corrosion resistance properties of C. hystrix wood. Genetic modification can be used to improve these properties.

Glycerol-bound oxidized fatty acids: formation and occurrence in peanuts

For peanuts, roasted at 170 °C, the formation of selected glycerol-bound oxidized fatty acids (GOFAs), namely 9-oxononanoic acid (9-ONA), azelaic acid (AZA) and octanoic acid, was observed by GC-MS (EI). The content of octanoic acid as well as AZA increased with continuous roasting time (from 59 mg/kg peanut oil to 101 mg/kg peanut oil and from not detectable to 8 mg/kg peanut oil, respectively), whereas the content of 9-ONA initially decreased from 25 mg/kg peanut oil to 8 mg/kg peanut oil (20 min) and increased again up to 37 mg/kg peanut oil following roasting for 40 min. Due to its aldehyde function, 9-ONA could contribute to amino acid side chain modifications as a result of lipation, which could directly influence the functional properties of peanut proteins. Both 9-ONA and octanoic acid are potential markers of thermal processes. Furthermore, in model experiments using methyl linoleate and methyl oleate, up to 18 oxidized fatty acids could be identified as methyl esters, 9-ONA as well as octanoic acid as major components and a faster formation of GOFAs under roasting conditions (170 °C, 20 min). In addition, 9-ONA contributes to the formation of AZA and octanoic acid in both free and bound form as a result of oxidative subsequent reactions in presence of iron (III).

Investigation of the changes in the lipid profiles in hairtail (Trichiurus haumela) muscle during frozen storage using chemical and LC/MS-based lipidomics analysis

Chemical and LC/MS-based lipidomics strategies were performed to explore the alterations of lipid profiles in hairtail (Trichiurus haumela) muscle during 120 days of frozen storage. Chemical results indicated that the PV and TBARS values in hairtail muscle significantly increased during 120 days of frozen storage. Lipidomics results detected 1223 lipids in hairtail muscle assigned to 26 lipid categories, including 261 triglycerides (TGs), 251 phosphatidylcholines (PCs), 153 phosphatidylethanolamines (PEs), and 66 diglycerides (DGs). Totally, 153 and 67 differentially abundant lipids (DALs) accumulated at high and low levels, respectively, were detected in frozen hairtail (FSH) compared to in fresh (FH) samples. Among these, PEs, PCs, and TGs/DGs as predominant lipid components were vulnerable to oxidation/hydrolysis mainly due to their unsaturated properties. The apparent alterations between FSH and FH samples may result from lipid side-chain modifications, backbone cleavage, and/or decomposition of lipids during long-term storage. This study provides novel insight into the molecular mechanisms of lipid alternations in hairtail muscle during frozen storage.

An Orthogonal Dynamic Covalent Chemistry Tool for Ring-Opening Polymerization of Cyclic Oligochalcogenides on Detachable Helical Peptide Templates.

A model system is introduced as a general tool to elaborate on orthogonal templation of dynamic covalent ring‐opening polymerization (ODC‐TROP). The tool consists of 310 helical peptides as unprecedented templates and semicarbazones as orthogonal dynamic covalent linkers. With difficult‐to‐control 1,2‐dithiolanes, ODC‐TROP on the level of short model oligomers occurs with high templation efficiency, increasing and diminishing upon helix stabilization and denaturation, respectively. Further, an anti‐templated conjugate with mispositioned monomers gave reduced templation upon helix twisting. Even with the “unpolymerizable” 1,2‐diselenolanes, initial studies already afford mild templation efficiency. These proof‐of‐principle results promise that the here introduced tool, recyclable and enabling late‐stage side chain modification, will be useful to realize ODC‐TROP of intractable or unknown cyclic dynamic covalent monomers for dynamer materials as well as cellular uptake and signaling applications.

Multi-reversible thermochromic polydiacetylene-CuZnFe2O4 magnetic nanocomposites with tunable colorimetric response to acid-base

Polydiacetylenes (PDAs) are materials that can be applied as colorimetric sensors for detecting a wide range of stimuli. The commercial PDA materials, constituting carboxylic headgroup, exhibit irreversible thermochromism and response to base. To extend the utilization, their sensing properties are often tuned by structural modification of PDA headgroup and/or side chain. In this study, we introduce a versatile method for fine tuning the properties of polydiacetylene (PDA) materials by incorporating CuZnFe2O4 magnetic nanoparticles. The resultant PDA/CuZnFe2O4 magnetic nanocomposites (MNCs) exhibit multi-reversible thermochromism with different shades of color transition. The MNCs also response to both acid and base. In addition, the sensitivity can be tuned by varying CuZnFe2O4 ratio. The PDA/CuZnFe2O4-MNCs are localizable via the application of magnetic field. These multifunctional MNCs show potential in various advanced technologies such as smart paints, anti-counterfeiting labels, electro-thermochromic displays and localizable acid/base sensors. The preparing process of MNCs is rather simple, making it suitable for industrial utilization.

WRKY33-mediated indolic glucosinolate metabolic pathway confers resistance against Alternaria brassicicola in Arabidopsis and Brassica crops.

The tryptophan (Trp)-derived plant secondary metabolites, including camalexin, 4-hydroxy-indole-3-carbonylnitrile, and indolic glucosinolate (IGS), show broad-spectrum antifungal activity. However, the distinct regulations of these metabolic pathways among different plant species in response to fungus infection are rarely studied. In this study, our results revealed that WRKY33 directly regulates IGS biosynthesis, notably the production of 4-methoxyindole-3-ylmethyl glucosinolate (4MI3G), conferring resistance to Alternaria brassicicola, an important pathogen which causes black spot in Brassica crops. WRKY33 directly activates the expression of CYP81F2, IGMT1, and IGMT2 to drive side-chain modification of indole-3-ylmethyl glucosinolate (I3G) to 4MI3G, in both Arabidopsis and Chinese kale (Brassica oleracea var. alboglabra Bailey). However, Chinese kale showed a more severe symptom than Arabidopsis when infected by Alternaria brassicicola. Comparative analyses of the origin and evolution of Trp metabolism indicate that the loss of camalexin biosynthesis in Brassica crops during evolution might attenuate the resistance of crops to Alternaria brassicicola. As a result, the IGS metabolic pathway mediated by WRKY33 becomes essential for Chinese kale to deter Alternaria brassicicola. Our results highlight the differential regulation of Trp-derived camalexin and IGS biosynthetic pathways in plant immunity between Arabidopsis and Brassica crops.

Correlation between molecular configuration and charge transfer dynamics in highly efficient organic solar cells

Molecular design and charge transport dynamics play an important role in achieving highly efficient organic solar cells (OSCs). In this work, non-fullerene acceptor Y6, its alkyl chain modified derivative N3 and halogen-replaced derivative BTP-BO-4Cl are selected to fabricate OSCs. The OSCs obtain a high power conversion efficiency (PCE) of 16.86%. The impact of different modification methods on molecular configuration and energy level matching is investigated systematically by time-resolved photoluminescence (TRPL) and transient absorption spectroscopy (TAS). The results demonstrate that side chain modification has a positive effect on molecular layer spacing, domain area size and charge carrier mobilities. More importantly, halogen atom replacing has the similar but more positive impact, bringing a higher PCE than side chain modification. The overall processes of charge generation, dissociation and recombination in OSCs are studied systematically for the first time. For the best-performance device, the lifetimes of singlet exciton recombination (τ1), charge transfer (CT) state recombination (τ2), singlet exciton dissociation (τ3) and CT state dissociation (τ4) are calculated as ∼500 ps, ∼2 ns, ∼200 fs and ∼1 ps, respectively. The correlation between molecular configuration and charge transfer dynamics is established, which can provide a guideline for designing materials of highly efficient OSCs.

Development of 6-Methanesulfonyl-8-nitrobenzothiazinone Based Antitubercular Agents.

The 6-trifluoro substituted 8-nitrobenzothiazinones (BTZs) represent a novel type of antitubercular agents, and their high antimycobacterial activity is related to the inhibition of decaprenylphosphoryl-β-d-ribose 2'-oxidase (DprE1), an enzyme essential for the biosynthesis of mycobacterial cell wall. While extraordinary whole-cell activity was reported for the clinically advanced compound PBTZ169, its poor aqueous solubility signals the potential low bioavailability. To ameliorate the BTZ physiochemical property, a series of 6-methanesulfonyl substituted compounds were designed and prepared, and their antitubercular activity and DprE1 inhibition ability were evaluated. Among these compounds, MsPBTZ169 and compounds 2 and 8 exhibited minimum inhibitory concentrations (MICs) of less than 40 nM; moreover, these compounds displayed increased aqueous solubility and acceptable metabolic stability. Taken together, this study suggested that the 6-methanesulfonyl substituted 8-nitrobenzothiazinone derivatives, in combination with side chain modification, might provide BTZ type antitubercular agents with improved drug-like properties.

Enhancement of the Thermoelectric Performance of n-Type Naphthalene Diimide-Based Conjugated Polymer by Engineering of Side Alkyl Chains

Developing n-type doped semiconducting polymers with high thermoelectric (TE) performance still remains challenging. In this paper, we show a new strategy to enhance the TE performance of n-doped naphthalene diimide (NDI)-based conjugated donor–acceptor (D-A) polymer by introducing one linear alkyl chain for each NDI unit. Film of the PNDI2T-1, in which each NDI unit is attached with one linear and one branching alkyl chains, exhibits higher electrical conductivity and a greater power factor (PF) than those of PNDI2T-2 with the same conjugated backbone and two branching side chains, after doping with either (4-(1,3-dimethyl-2,3-dihydro-1H-benzoimidazol-2-yl)phenyl)dimethylamine (N-DMBI) or decahydro-3a,6a,9a-triazaphenalene (TAM) under the same conditions. The optimal PF values of the doped films of PNDI2T-1 with N-DMBI and TAM can reach 1.6 and 3.5 μW m–1K–2, respectively, which are among the highest reported TE performances for NDI-based semiconducting polymers after n-doping. The higher TE performance of the doped films of PNDI2T-1 is attributed to the enhancement of charge mobility and improvement of doping degree after side-chain modification. These results provide a new molecular design rationale for the future development of high-performance n-type thermoelectric semiconducting polymers.

Polymerized Small Molecular Acceptor with Branched Side Chains for All Polymer Solar Cells with Efficiency over 16.7%

The power conversion efficiencies (PCEs) of small molecule acceptor (SMA)-based organic solar cells have already exceeded 18%. However, the development of polymer acceptors still lags far behind their SMA counterparts mainly due to the lack of efficient polymer acceptors. Herein, a series of polymer acceptors named PY-X (with X being the branched alkyl chain) aredesigned and synthesized by employing the same central core with the SMA L8-BO but with different branched alkyl chains on the pyrrole motif. It is found that the molecular packing of SMA-HD featuring 2-hexyldecyl side chain used in the synthesis of PY-HD is similar to L8-BO, in which the branched alkyl chains lead to condensed and high-order molecular assembly in SMA-HD molecules. When combined with PM6, PY-HD-based all polymer solar cell (all-PSC) exhibits a high PCE of 16.41%, representing the highest efficiency for the binary all-PSCs. Moreover, the side-chain modification on the pyrrole site position further improves the performance of the all-PSCs, and the PY-DT-based device delivers a new record high efficiency of 16.76% (certified as 16.3%). The work provides new insights for understanding the structure-property relationship of polymer acceptors and paves a feasible avenue to develop efficient conjugated polymer acceptors.

Performances of two side-chain modified medium-bandgap alternating polymers with main-chain twisted non-fullerene acceptor

Two alternating polymers (P3 and P4) based on side-chain modified benzo[1,2-b:4,5-b′]dithiophene as the electron-donating unit and fluorinated benzothiadiazole derivatives as the electron-withdrawing segment were adopted as donor materials to blend with a main-chain twisted non-fullerene acceptor (i-IEICO-4 F) for bulk heterojunction polymer solar cells (PSCs). The effects of the two types of side-chain substitutions on the UV-Vis absorptive characteristics, frontier molecular orbitals, and photovoltaic performances of the polymers were systematically probed. Both two polymers have medium-bandgaps, 1.75 eV for P3 and 1.71 eV for P4, respectively, guaranteeing the complementary absorption with i-IEICO-4 F. Among these polymers, the device of polymer P3, which has 2,3-octylthienyl side-chain, shows enhanced dielectric constant, suppressed trap-assisted recombination, promoted hole mobility with more balanced carrier mobility, and suitable phase separation, thus successfully achieving an efficiency of 10.67%. While the alkoxylphenyl substituted polymer P4 has slightly deepened molecular energy level and bathochromic-shifted absorption spectrum and achieved enhanced open-circuit voltage, improved electron mobility, and inhibitive bimolecular recombination in the device, thus acquiring a slightly reduced efficiency of 10.19%. This work not only elaborates the applicability of benzothiadiazole-based medium bandgap polymer donors with main-chain twisted acceptors but also reveals the importance of the side-chain modification of the polymer donors on the performances of the fabricated PSCs.

Structure and Function of the β-Asp-Arg Polymerase Cyanophycin Synthetase 2.

Cyanophycin is a biopolymer composed of long chains of β-Asp-Arg. It is widespread in nature, being synthesized by many clades of bacteria, which use it as a cellular reservoir of nitrogen, carbon, and energy. Two enzymes are known to produce cyanophycin: cyanophycin synthetase 1 (CphA1), which builds cyanophycin from the amino acids Asp and Arg by alternating between two separate reactions for backbone extension and side chain modification, and cyanophycin synthetase 2 (CphA2), which polymerizes β-Asp-Arg dipeptides. CphA2 is evolutionarily related to CphA1, but questions about CphA2's altered structure and function remain unresolved. Cyanophycin and related molecules have drawn interest as green biopolymers. Because it only has a single active site, CphA2 could be more useful than CphA1 for biotechnological applications seeking to produce modified cyanophycin. In this study, we report biochemical assays on nine cyanobacterial CphA2 enzymes and report the crystal structure of CphA2 from Gloeothece citriformis at 3.0 Å resolution. The structure reveals a homodimeric, three-domain architecture. One domain harbors the polymerization active site and the two other domains have structural roles. The structure and biochemical assays explain how CphA2 binds and polymerizes β-Asp-Arg and highlights differences in in vitro oligomerization and activity between CphA2 enzymes. Using the structure and distinct activity profile as a guide, we introduced a single point mutation that converted Gloeothece citriformis CphA2 from a primer-dependent enzyme into a primer-independent enzyme.