MTHFD2-mediated redox homeostasis promotes gastric cancer progression under hypoxic conditions.

OBJECTIVES: Cancer cells undergo metabolic reprogramming to adapt to high oxidative stress, but little is known about how metabolic remodeling enables gastric cancer cells to survive stress associated with aberrant reactive oxygen species (ROS) production. Here, we aimed to identify the key metabolic enzymes that protect gastric cancer (GC) cells from oxidative stress. METHODS: ROS level was detected by DCFH-DA probes. Multiple cell biological studies were performed to identify the underlying mechanisms. Furthermore, cell-based xenograft and patient-derived xenograft (PDX) model were performed to evaluate the role of MTHFD2 in vivo. RESULTS: We found that overexpression of MTHFD2, but not MTHFD1, is associated with reduced overall and disease-free survival in gastric cancer. In addition, MTHFD2 knockdown reduces the cellular NADPH/NADP+ ratio, colony formation and mitochondrial function, increases cellular ROS and cleaved PARP levels and induces in cell death under hypoxia, a hallmark of solid cancers and a common inducer of oxidative stress. Moreover, genetic or pharmacological inhibition of MTHFD2 reduces tumor burden in both tumor cell lines and patient-derived xenograft-based models. DISCUSSION: our study highlights the crucial role of MTHFD2 in redox regulation and tumor progression, demonstrating the therapeutic potential of targeting MTHFD2.

Maximizing the potential of nitrilase: Unveiling their diversity, catalytic proficiency, and versatile applications.

Nitrilases represent a distinct class of enzymes that play a pivotal role in catalyzing the hydrolysis of nitrile compounds, leading to the formation of corresponding carboxylic acids. These enzymatic entities have garnered significant attention across a spectrum of industries, encompassing pharmaceuticals, agrochemicals, and fine chemicals. Moreover, their significance has been accentuated by mounting environmental pressures, propelling them into the forefront of biodegradation and bioremediation endeavors. Nevertheless, the natural nitrilases exhibit intrinsic limitations such as low thermal stability, narrow substrate selectivity, and inadaptability to varying environmental conditions. In the past decade, substantial efforts have been made in elucidating the structural underpinnings and catalytic mechanisms of nitrilase, providing basis for engineering of nitrilases. Significant breakthroughs have been made in the regulation of nitrilases with ideal catalytic properties and application of the enzymes for industrial productions. This review endeavors to provide a comprehensive discourse and summary of recent research advancements related to nitrilases, with a particular emphasis on the elucidation of the structural attributes, catalytic mechanisms, catalytic characteristics, and strategies for improving catalytic performance of nitrilases. Moreover, the exploration extends to the domain of process engineering and the multifarious applications of nitrilases. Furthermore, the future development trend of nitrilases is prospected, providing important guidance for research and application in the related fields.

Genomic Insights into Cyanide Biodegradation in the Pseudomonas Genus.

Molecular studies about cyanide biodegradation have been mainly focused on the hydrolytic pathways catalyzed by the cyanide dihydratase CynD or the nitrilase NitC. In some Pseudomonas strains, the assimilation of cyanide has been linked to NitC, such as the cyanotrophic model strain Pseudomonas pseudoalcaligenes CECT 5344, which has been recently reclassified as Pseudomonas oleovorans CECT 5344. In this work, a phylogenomic approach established a more precise taxonomic position of the strain CECT 5344 within the species P. oleovorans. Furthermore, a pan-genomic analysis of P. oleovorans and other species with cyanotrophic strains, such as P. fluorescens and P. monteilii, allowed for the comparison and identification of the cioAB and mqoAB genes involved in cyanide resistance, and the nitC and cynS genes required for the assimilation of cyanide or cyanate, respectively. While cyanide resistance genes presented a high frequency among the analyzed genomes, genes responsible for cyanide or cyanate assimilation were identified in a considerably lower proportion. According to the results obtained in this work, an in silico approach based on a comparative genomic approach can be considered as an agile strategy for the bioprospection of putative cyanotrophic bacteria and for the identification of new genes putatively involved in cyanide biodegradation.

Cryo-EM structure of bacterial nitrilase reveals insight into oligomerization, substrate recognition, and catalysis.

Many enzymes can self-assemble into higher-order structures with helical symmetry. A particularly noteworthy example is that of nitrilases, enzymes in which oligomerization of dimers into spiral homo-oligomers is a requirement for their enzymatic function. Nitrilases are widespread in nature where they catalyze the hydrolysis of nitriles into the corresponding carboxylic acid and ammonia. Here, we present the Cryo-EM structure, at 3 Å resolution, of a C-terminal truncate nitrilase from Rhodococcus sp. V51B that assembles in helical filaments. The model comprises a complete turn of the helical arrangement with a substrate-intermediate bound to the catalytic cysteine. The structure was solved having added the substrate to the protein. The length and stability of filaments was made more substantial in the presence of the aromatic substrate, benzonitrile, but not for aliphatic nitriles or dinitriles. The overall structure maintains the topology of the nitrilase family, and the filament is formed by the association of dimers in a chain-like mechanism that stabilizes the spiral. The active site is completely buried inside each monomer, while the substrate binding pocket was observed within the oligomerization interfaces. The present structure is in a closed configuration, judging by the position of the lid, suggesting that the intermediate is one of the covalent adducts. The proximity of the active site to the dimerization and oligomerization interfaces, allows the dimer to sense structural changes once the benzonitrile was bound, and translated to the rest of the filament, stabilizing the helical structure.

Multiplexed in-situ mutagenesis driven by a dCas12a-based dual-function base editor.

Mutagenesis driving genetic diversity is vital for understanding and engineering biological systems. However, the lack of effective methods to generate in-situ mutagenesis in multiple genomic loci combinatorially limits the study of complex biological functions. Here, we design and construct MultiduBE, a dCas12a-based multiplexed dual-function base editor, in an all-in-one plasmid for performing combinatorial in-situ mutagenesis. Two synthetic effectors, duBE-1a and duBE-2b, are created by amalgamating the functionalities of cytosine deaminase (from hAPOBEC3A or hAID*Δ ), adenine deaminase (from TadA9), and crRNA array processing (from dCas12a). Furthermore, introducing the synthetic separator Sp4 minimizes interference in the crRNA array, thereby facilitating multiplexed in-situ mutagenesis in both Escherichia coli and Bacillus subtilis. Guided by the corresponding crRNA arrays, MultiduBE is successfully employed for cell physiology reprogramming and metabolic regulation. A novel mutation conferring streptomycin resistance has been identified in B. subtilis and incorporated into the mutant strains with multiple antibiotic resistance. Moreover, surfactin and riboflavin titers of the combinatorially mutant strains improved by 42% and 15-fold, respectively, compared with the control strains with single gene mutation. Overall, MultiduBE provides a convenient and efficient way to perform multiplexed in-situ mutagenesis.

Modification of nitrilase based on computer screening and efficient biosynthesis of 4-cyanobenzoic acid.

4-cyanobenzoic acid serves as a crucial intermediate for the synthesis of various high-value organic compounds. The enzymatic hydrolysis of terephthalonitrile to produce 4-cyanobenzoic acid using nitrilase offers the advantages of a simple reaction pathway, environmental friendliness, and easy product separation. In order to efficiently develop nitrilases that meet industrial production requirements, the virtual screening method used in the study is established and mature. From a total of 371 amino acids in the nitrilase AfNIT, which exhibits activity in terephthalonitrile hydrolysis, three candidate sites (F168, S192, and T201) were identified, and a "small and accurate" mutant library was constructed. The triple mutant F168V/T201N/S192F was screened from this small mutant library with a specific activity of 227.3 U mg-1 , which was 3.8 times higher than that of the wild-type AfNIT. Using the whole-cell biocatalyst containing the mutant F168V/T201N/S192F, terephthalonitrile was successfully hydrolyzed at a concentration of 150 g L-1 to produce 4-cyanobenzoic acid with a final yield of 170.3 g L-1 and a conversion rate of 98.7%. The obtained nitrilase mutant F168V/T201N/S192F in this study can be effectively applied in the biomanufacturing of 4-cyanobenzoic acid using terephthalonitrile as a substrate. Furthermore, the results also demonstrate the significant improvement in predictive accuracy achieved through the latest AI-assisted computer simulation methods. This approach represents a promising and feasible new technological pathway for assisting enzyme engineering research, laying a theoretical foundation for other related studies.

Interference with MTHFD2 induces ferroptosis in ovarian cancer cells through ERK signaling to suppress tumor malignant progression.

Ovarian cancer (OC) is a deadliest gynecological cancer with the highest mortality rate. Methylenetetrahydrofolate dehydrogenase 2 (MTHFD2), a crucial tumor-promoting factor, is over-expressed in several malignancies including OC. The present study aimed to explore the role and mechanisms of MTHFD2 in OC malignant progression. Thus, cell proliferation, cycling, apoptosis, migration, and invasion were evaluated by CCK-8 assay, EdU assay, flow cytometry, wound healing, transwell assay and western blotting. Additionally, glycolysis was assessed by measuring the level of glucose and lactate production, as well as the expressions of GLUT1, HK2 and PKM2. Then the expression of ferroptosis-related proteins and ERK signaling was detected using western blotting. Ferroptosis was detected through the measurement of iron level, GSH, MDA and ROS activities. The results revealed that MTHFD2 was highly expressed in OC cells. Besides, interference with MTHFD2 induced ferroptosis, promoted ROS accumulation, destroyed mitochondrial function, reduced ATP content and inhibited glycolysis in OC cells. Subsequently, we further found that interference with MTHFD2 affected mitochondrial function and glycolysis in OC cells through ERK signaling. Moreover, interference with MTHFD2 affected ferroptosis to inhibit the malignant progression of OC cells. Collectively, our present study disclosed that interference with MTHFD2 induced ferroptosis in OC to inhibit tumor malignant progression through regulating ERK signaling.

Bi-allelic NIT1 variants cause a brain small vessel disease characterized by movement disorders, massively dilated perivascular spaces, and intracerebral hemorrhage.

PURPOSE: To describe a recessively inherited cerebral small vessel disease, caused by loss-of-function variants in Nitrilase1 (NIT1). METHODS: We performed exome sequencing, brain magnetic resonance imaging, neuropathology, electron microscopy, western blotting, and transcriptomic and metabolic analyses in 7 NIT1-small vessel disease patients from 5 unrelated pedigrees. RESULTS: The first identified patients were 3 siblings, compound heterozygous for the NIT1 c.727C>T; (p.Arg243Trp) variant and the NIT1 c.198_199del; p.(Ala68∗) variant. The 4 additional patients were single cases from 4 unrelated pedigrees and were all homozygous for the NIT1 c.727C>T; p.(Arg243Trp) variant. Patients presented in mid-adulthood with movement disorders. All patients had striking abnormalities on brain magnetic resonance imaging, with numerous and massively dilated basal ganglia perivascular spaces. Three patients had non-lobar intracerebral hemorrhage between age 45 and 60, which was fatal in 2 cases. Western blotting on patient fibroblasts showed absence of NIT1 protein, and metabolic analysis in urine confirmed loss of NIT1 enzymatic function. Brain autopsy revealed large electron-dense deposits in the vessel walls of small and medium sized cerebral arteries. CONCLUSION: NIT1-small vessel disease is a novel, autosomal recessively inherited cerebral small vessel disease characterized by a triad of movement disorders, massively dilated basal ganglia perivascular spaces, and intracerebral hemorrhage.

Protein Interaction Map of APOBEC3 Enzyme Family Reveals Deamination-Independent Role in Cellular Function.

Human APOBEC3 enzymes are a family of single-stranded (ss)DNA and RNA cytidine deaminases that act as part of the intrinsic immunity against viruses and retroelements. These enzymes deaminate cytosine to form uracil which can functionally inactivate or cause degradation of viral or retroelement genomes. In addition, APOBEC3s have deamination-independent antiviral activity through protein and nucleic acid interactions. If expression levels are misregulated, some APOBEC3 enzymes can access the human genome leading to deamination and mutagenesis, contributing to cancer initiation and evolution. While APOBEC3 enzymes are known to interact with large ribonucleoprotein complexes, the function and RNA dependence are not entirely understood. To further understand their cellular roles, we determined by affinity purification mass spectrometry (AP-MS) the protein interaction network for the human APOBEC3 enzymes and mapped a diverse set of protein-protein and protein-RNA mediated interactions. Our analysis identified novel RNA-mediated interactions between APOBEC3C, APOBEC3H Haplotype I and II, and APOBEC3G with spliceosome proteins, and APOBEC3G and APOBEC3H Haplotype I with proteins involved in tRNA methylation and ncRNA export from the nucleus. In addition, we identified RNA-independent protein-protein interactions with APOBEC3B, APOBEC3D, and APOBEC3F and the prefoldin family of protein-folding chaperones. Interaction between prefoldin 5 (PFD5) and APOBEC3B disrupted the ability of PFD5 to induce degradation of the oncogene cMyc, implicating the APOBEC3B protein interaction network in cancer. Altogether, the results uncover novel functions and interactions of the APOBEC3 family and suggest they may have fundamental roles in cellular RNA biology, their protein-protein interactions are not redundant, and there are protein-protein interactions with tumor suppressors, suggesting a role in cancer biology. Data are available via ProteomeXchange with the identifier PXD044275.

Identification and evolution of non-traditional nitrilase from Spirosoma linguale DSM 74 with high hydration activity.

Hydration, a secondary activity mediated by nitrilase, is a promising new pathway for amide production. However, low hydration activity of nitrilase or trade-off between hydration and catalytic activity hinders its application in the production of amides. Here, natural C-terminal-truncated wild-type nitrilase, mined from a public database, obtained a high-hydration activity nitrilase as a novel evolutionary starting point for further protein engineering. The nitrilase Nit-74 from Spirosoma linguale DSM 74 was successfully obtained and exhibited the highest hydration activity level, performing 50.7 % nicotinamide formation and 87.6 % conversion to 2 mM substrate 3-cyanopyridine. Steric hindrance of the catalytic activity center and the N-terminus of the catalytic cysteine residue helped us identify three key residues: I166, W168, and T191. Saturation mutations resulted in three single mutants that further improved the hydration activity of N-heterocyclic nitriles. Among them, the mutant T191S performed 72.7 % nicotinamide formation, which was much higher than the previously reported highest level of 18.7 %. Additionally, mutants I166N and W168Y exhibited a 97.5 % 2-picolinamide ratio and 97.7 % isonicotinamide ratio without any loss of catalytic activity, which did not indicate a trade-off effect. Our results expand the screening and evolution library of promiscuous nitrilases with high hydration activity for amide formation.

Cloning and heterologous expression of Fusarium oxysporum nitrilase gene in Escherichia coli and evaluation in cyanide degradation.

Cyanide is widely utilized in the extraction of precious metal extraction even though it has been deemed as the most toxic compound. Fusarium oxysporum has been shown to degrade cyanide through the activity of the Nitrilase enzyme. In this study, the coding sequence of nitrilase gene from F. oxysporum genomic DNA was optimized for cloning and expression in E. coli. The pUC57 containing synthetic optimized nitrilase gene was transferred into E. coli DH5α strain. This nitrilase gene was sub-cloned into pET26b (+) expression vector containing an in-built His-tag at the C-terminal end to facilitate its purification. The recombinant plasmid, pETAM1, was confirmed by PCR, digestion pattern, and sequencing. The recombinant protein was overproduced in E. coli BL21 (DE3). The results of the SDS-PAGE pattern and Western blot analysis confirmed the expression of the expected recombinant protein. For expression optimization of Nitrilase protein, M16 orthogonal experimental design of the Taguchi method was used. The effect of induction time, temperature and IPTG concentration were examined using four levels for each factors. Estimation of the amount of the expressed protein was calculated via densitometry on SDS-PAGE. The enzyme activity and expression in E. coli proved to be successful since there was ammonia production when potassium cyanide and acrylonitrile were used as substrates while the highest enzyme activity of 88% was expressed at 30 °C. The Km and Vm values of the expressed Nitrilase enzyme were determined to be 0.68 mM and 0.48 mM/min respectively.

Construing recombinant ZFP160 from Aspergillus terreus as pterin deaminase enzyme.

Pterin deaminase stands as a metalloenzyme and exhibits both antitumor and anticancer activities. Therefore, this study aimed to explore the molecular function of zinc finger protein-160 (zfp160) from Aspergillus terreus with its enzyme mechanism in detail. Subsequently, preliminary molecular docking studies on zfp160 from A. terreus were done. Next, the cloning and expression of zfp160 protein were carried out. Following, protein expression was induced and purified through nickel NTA column with imidazole gradient elution. Through the Mascot search engine tool, the expressed protein of MALDI-TOF was confirmed by 32 kDa bands of SDS-PAGE. Furthermore, its enzymatic characterization and biochemical categorization were also explored. The optimum conditions for enzyme were determined to be pH 8, temperature 35°C, km 50 µm with folic acid as substrate, and Vmax of 24.16 (IU/mL). Further, in silico analysis tried to explore the interactions and binding affinity of various substrates to the modeled pterin deaminase from A. terreus. Our results revealed the binding mode of different substrate molecules with pterin deaminase using the approximate scoring functions that possibly correlate with actual experimental binding affinities. Following this, molecular dynamic simulations provided the in-depth knowledge on deciphering functional mechanisms of pterin deaminase over other drugs.

Structural basis of HIV-1 Vif-mediated E3 ligase targeting of host APOBEC3H.

Human APOBEC3 (A3) cytidine deaminases are antiviral factors that are particularly potent against retroviruses. As a countermeasure, HIV-1 uses a viral infectivity factor (Vif) to target specific human A3s for proteasomal degradation. Vif recruits cellular transcription cofactor CBF-ß and Cullin-5 (CUL5) RING E3 ubiquitin ligase to bind different A3s distinctively, but how this is accomplished remains unclear in the absence of the atomic structure of the complex. Here, we present the cryo-EM structures of HIV-1 Vif in complex with human A3H, CBF-ß and components of CUL5 ubiquitin ligase (CUL5, ELOB, and ELOC). Vif nucleates the entire complex by directly binding four human proteins, A3H, CBF-ß, CUL5, and ELOC. The structures reveal a large interface area between A3H and Vif, primarily mediated by an α-helical side of A3H and a five-stranded ß-sheet of Vif. This A3H-Vif interface unveils the basis for sensitivity-modulating polymorphism of both proteins, including a previously reported gain-of-function mutation in Vif isolated from HIV/AIDS patients. Our structural and functional results provide insights into the remarkable interplay between HIV and humans and would inform development efforts for anti-HIV therapeutics.

Quercitrin alleviates lipid metabolism disorder in polycystic ovary syndrome-insulin resistance by upregulating PM20D1 in the PI3K/Akt pathway.

BACKGROUND: Abnormal endocrine metabolism caused by polycystic ovary syndrome combined with insulin resistance (PCOS-IR) poses a serious risk to reproductive health in females. Quercitrin is a flavonoid that can efficiently improve both endocrine and metabolic abnormalities. However, it remains unclear if this agent can exert therapeutic effect on PCOS-IR. METHODS: The present study used a combination of metabolomic and bioinformatic methods to screen key molecules and pathways involved in PCOS-IR. A rat model of PCOS-IR and an adipocyte IR model were generated to investigate the role of quercitrin in regulating reproductive endocrine and lipid metabolism processes in PCOS-IR. RESULTS: Peptidase M20 domain containing 1 (PM20D1) was screened using bioinformatics to evaluate its participation in PCOS-IR. PCOS-IR regulation via the PI3K/Akt signaling pathway was also investigated. Experimental analysis showed that PM20D1 levels were reduced in insulin-resistant 3T3-L1 cells and a letrozole PCOS-IR rat model. Reproductive function was inhibited, and endocrine metabolism was abnormal. The loss of adipocyte PM20D1 aggravated IR. In addition, PM20D1 and PI3K interacted with each other in the PCOS-IR model. Furthermore, the PI3K/Akt signaling pathway was shown to participate in lipid metabolism disorders and PCOS-IR regulation. Quercitrin reversed these reproductive and metabolic disorders. CONCLUSION: PM20D1 and PI3K/Akt were required for lipolysis and endocrine regulation in PCOS-IR to restore ovarian function and maintain normal endocrine metabolism. By upregulating the expression of PM20D1, quercitrin activated the PI3K/Akt signaling pathway, improved adipocyte catabolism, corrected reproductive and metabolic abnormalities, and had a therapeutic effect on PCOS-IR.

Geometric Remodeling of Nitrilase Active Pocket Based on ALF-Scanning Strategy To Enhance Aromatic Nitrile Substrate Preference and Catalytic Efficiency.

Nitrilase can catalyze nitrile compounds to generate corresponding carboxylic acids. Nitrilases as promiscuous enzymes can catalyze a variety of nitrile substrates, such as aliphatic nitriles, aromatic nitriles, etc. However, researchers tend to prefer enzymes with high substrate specificity and high catalytic efficiency. In this study, we developed an active pocket remodeling (ALF-scanning) based on modulating the geometry of the nitrilase active pocket to alter substrate preference and improve catalytic efficiency. Using this strategy, combined with site-directed saturation mutagenesis, we successfully obtained 4 mutants with strong aromatic nitrile preference and high catalytic activity, W170G, V198L, M197F, and F202M, respectively. To explore the synergistic relationship of these 4 mutations, we constructed 6 double-combination mutants and 4 triple-combination mutants. By combining mutations, we obtained the synergistically enhanced mutant V198L/W170G, which has a significant preference for aromatic nitrile substrates. Compared with the wild type, its specific activities for 4 aromatic nitrile substrates are increased to 11.10-, 12.10-, 26.25-, and 2.55-fold, respectively. By mechanistic dissection, we found that V198L/W170G introduced a stronger substrate-residue π-alkyl interaction in the active pocket and obtained a larger substrate cavity (225.66 Å3 to 307.58 Å3), making aromatic nitrile substrates more accessible to be catalyzed by the active center. Finally, we conducted experiments to rationally design the substrate preference of 3 other nitrilases based on the substrate preference mechanism and also obtained the corresponding aromatic nitrile substrate preference mutants of these three nitrilases and these mutants with greatly improved catalytic efficiency. Notably, the substrate range of SmNit is widened. IMPORTANCE In this study, the active pocket was largely remodeled based on the ALF-scanning strategy we developed. It is believed that ALF-scanning not only could be employed for substrate preference modification but might also play a role in protein engineering of other enzymatic properties, such as substrate region selectivity and substrate spectrum. In addition, the mechanism of aromatic nitrile substrate adaptation we found is widely applicable to other nitrilases in nature. To a large extent, it could provide a theoretical basis for the rational design of other industrial enzymes.

The application of adenine deaminase in antibody affinity maturation.

Previously, we established a platform for antibody/protein affinity maturation based on CHO cell display. The gene of interest was mutated by activation-induced cytidine deaminase (AID), and then, a mutation library mainly containing G/C to A/T conversion was obtained by simply proliferating cells. However, the AID-induced G/C to A/T conversion limits the diversity space of the mutation library. In contrast to AID, adenine deaminase (ADA) can convert A/T to G/C. In this study, we demonstrated that ADA could efficiently induce random A/T to G/C mutations on the target gene in the CHO cell display and could be applied in affinity maturation. Our data also showed that more mutant types were obtained through the combined use of AID and ADA, thus offering an opportunity to acquire new mutants offering higher affinities than those obtained by only using AID. Examples presented in this study showed that ADA contributed to the improvement of antibody affinity either with or without AID in CHO display. KEY POINTS: • ADA is able to induce random mutations on antibody gene in mammalian cells. • ADA induces mutations on A/T bases to compensate AID which can induce mutation on G/C. • Combination of AID and ADA can increase mutation types and maturation efficiencies.

Stability of APOBEC3F in the Presence of the APOBEC3 Antagonist HIV-1 Vif Increases at the Expense of Co-Expressed APOBEC3H Haplotype I.

The seven human APOBEC3 enzymes (APOBEC3A through H, excluding E) are host restriction factors. Most of the APOBEC3 enzymes can restrict HIV-1 replication with different efficiencies. The HIV-1 Vif protein combats APOBEC3-mediated restriction by inducing ubiquitination and degradation in the proteasome. APOBEC3F and APOBEC3G can hetero-oligomerize, which increases their restriction capacity and resistance to Vif. Here we determined if APOBEC3C, APOBEC3F, or APOBEC3G could hetero-oligomerize with APOBEC3H haplotype I. APOBEC3H haplotype I has a short half-life in cells due to ubiquitination and degradation by host proteins, but is also resistant to Vif. We hypothesized that hetero-oligomerization with APOBEC3H haplotype I may result in less Vif-mediated degradation of the interacting APOBEC3 and stabilize APOBEC3H haplotype I, resulting in more efficient HIV-1 restriction. Although we found that all three APOBEC3s could interact with APOBEC3H haplotype I, only APOBEC3F affected APOBEC3H haplotype I by surprisingly accelerating its proteasomal degradation. However, this increased APOBEC3F levels in cells and virions in the absence or presence of Vif and enabled APOBEC3F-mediated restriction of HIV-1 in the presence of Vif. Altogether, the data suggest that APOBEC3 enzymes can co-regulate each other at the protein level and that they cooperate to ensure HIV-1 inactivation rather than evolution.

Identification and structural prediction of the unrevealed amidohydrolase enzyme: Pterin deaminase from Agrobacterium tumefaciens LBA4404.

Microbes make a remarkable contribution to the health and well-being of living beings all over the world. Interestingly, pterin deaminase is an amidohydrolase enzyme that exhibits antitumor, anticancer activities and antioxidant properties. With the existing evidence of the presence of pterin deaminase from microbial sources, an attempt was made to reveal the existence of this enzyme in the unexplored bacterium Agrobacterium tumefaciens LBA4404. After, the cells were harvested and characterized as intracellular enzymes and then partially purified through acetone precipitation. Subsequently, further purification step was carried out with an ion-exchange chromatogram (HiTrap Q FF) using the Fast-Protein Liquid Chromatography technique (FPLC). Henceforward, the approximate molecular weight of the purified pterin deaminase was determined through SDS-PAGE. Furthermore, the purified protein was identified accurately by MALDI-TOF, and the sequence was explored through a Mascot search engine. Additionally, the three-dimensional structure was predicted and then validated, as well as ligand-binding sites, and the stability of this enzyme was confirmed for the first time. Thus, the present study revealed the selected parameters showing a considerable impact on the identification and purification of pterin deaminase from A. tumefaciens LBA4404 for the first time. The enzyme specificity makes it a favorable choice as a potent anticancer agent.

Biotransformation of 3-cyanopyridine to nicotinic acid using whole-cell nitrilase of Gordonia terrae mutant MN12.

In the present study, the Gordonia terrae was subjected to chemical mutagenesis using ethyl methane sulfonate (EMS) and methyl methane sulfonate (MMS), N-methyl-N-nitro-N-nitrosoguanidine (MNNG), 5-bromouracil (5-BU) and hydroxylamine with the aim of improving the catalytic efficiency of its nitrilase for conversion of 3-cyanopyridine to nicotinic acid. A mutant MN12 generated with MNNG exhibited increase in nitrilase activity from 0.5 U/mg dcw (dry cell weight) (in the wild G. terrae) to 1.33 U/mg dcw. Further optimizations of culture conditions using response surface methodology enhanced the enzyme production to 1.2-fold. Whole-cell catalysis was adopted for bench-scale synthesis of nicotinic acid, and 100% conversion of 100 mM 3-cyanopyridine was achieved in potassium phosphate buffer (0.1 M, pH 8.0) at 40 °C in 15 min. The whole-cell nitrilase of the mutant MN12 exhibited higher rate of product formation and volumetric productivity, i.e., 24.56 g/h/g dcw and 221 g/L as compared to 8.95 g/h/g dcw and 196.8 g/L of the wild G. terrae. The recovered product was confirmed by HPLC, FTIR and NMR analysis with high purity (> 99.9%). These results indicated that the mutant MN12 of G. terrae as whole-cell nitrilase is a very promising biocatalyst for the large-scale synthesis of nicotinic acid.

Green synthesis aspects of (R)-(-)-mandelic acid; a potent pharmaceutically active agent and its future prospects.

(R)-(-)-mandelic acid is an important carboxylic acid known for its numerous potential applications in the pharmaceutical industry as it is an ideal starting material for the synthesis of antibiotics, antiobesity drugs and antitumor agents. In past few decades, the synthesis of (R)-(-)-mandelic acid has been undertaken mainly through the chemical route. However, chemical synthesis of optically pure (R)-(-)-mandelic acid is difficult to achieve at an industrial scale. Therefore, its microbe mediated production has gained considerable attention as it exhibits many merits over the chemical approaches. The present review focuses on various biotechnological strategies for the production of (R)-(-)-mandelic acid through microbial biotransformation and enzymatic catalysis; in particular, an analysis and comparison of the synthetic methods and different enzymes. The wild type as well as recombinant microbial strains for the production of (R)-(-)-mandelic acid have been elucidated. In addition, different microbial strategies used for maximum bioconversion of mandelonitrile into (R)-(-)-mandelic acid are discussed in detail with regard to higher substrate tolerance and maximum bioconversion.HighlightsMandelonitrile, mandelamide and o-chloromandelonitrile can be used as substrates to produce (R)-(-)-mandelic acid by enzymes.Three enzymes (nitrilase, nitrile hydratase and amidase) are systematically introduced for production of (R)-(-)-mandelic acid.Microbial transformation is able to produce optically pure (R)-(-)-mandelic acid with 100% productive yield.