BIOTECHNOLOGY AND APPLIED BIOCHEMISTRY
·
2023
·
IF 2.8
Shijin Wu,Qiang Wang,Xiaojing Ma,Lequan Qiu,Hongde Yan
The amino acid residues at the entrance of the catalytic pocket may impose steric hindrance on the substrate to enter the active center of the enzyme. Based on the analysis of the three-dimensional structure of the Saccharomyces cerevisiae old yellow enzyme 3 (OYE3), four bulky residues were chosen and mutated to small amino acids. The results showed that mutation of the W116 residue had interesting impacts on the catalytic performance. All four variants became inactive for the reduction of ( R )-carvone and ( S )-carvone, but inverted the stereoselectivity for the reduction of ( E / Z )-citral. The mutation of the F250 residue had a more positive effect on the activity and stereoselectivity. Two variants, F250A and F250S, showed excellent diastereoselectivity and activity for the reduction of ( R )-carvone (de > 99%, c > 99%) and increased diastereoselectivity and activity for the reduction of ( S )-carvone (de > 96%, c > 80%). One variant of the P295 residue, P295G, displayed excellent diastereoselectivity and activity only for the reduction of ( R )-carvone (de > 99%, c > 99%). Mutation of the Y375 residue had a negative impact on the activity of the enzyme. These findings provide some solutions for rational enzyme engineering of OYE3.
研究领域:
生物化学
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酶学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
ECOTOXICOLOGY AND ENVIRONMENTAL SAFETY
·
2023
·
IF 6.8
Jiali Li,Linglong Dai,Yu Feng,Zhenxiao Cao,Yuting Ding,Hao Xu,An Xu,Hua Du
Flame retardants (FRs) have raised public concerns because of their environmental persistence and negative impacts on human health. Recent evidence has revealed that many FRs exhibit reproductive toxicities and transgenerational impacts, whereas the toxic effects of FRs on germ cells remain barely explored. Here we investigated the multigenerational effects of three flame retardants (TBBPA, TCEP and TCPP) on germ cell development in Caenorhabditis elegans , and examined the germ cell mutagenicity of these FRs by using whole genome sequencing. Parental exposure to three FRs markedly increased germ cell apoptosis, and impeded oogenesis in F1-F6 offspring. In addition, the double-increased mutation frequencies observed in progeny genomes uncover the mutagenic actions of FRs on germ cells. Analysis of mutation spectra revealed that these FRs predominantly induced point mutations at A:T base pairs, whereas both small and large indels were almost unaffected. These results revealed the long-term effects of FRs on development and genomic stability of germ cells, which may pose risks to environmental organisms and human reproductive health. Taken together, our findings suggest that germ cell mutagenicity should be carefully examined for the environmental risk assessment of FRs and other emerging pollutants.
研究领域:
环境科学
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遗传学
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毒理学
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产品关联
DOI
PubMed
高影响因子
Molecular Catalysis
·
2022
·
IF 5.089
Jiacheng Zhang,Jieyu Zhou,Guochao Xu,Ye Ni
Kp ADH is a promising biocatalyst for the synthesis of chiral bulky-bulky alcohols. Substrate specificity toward diaryl ketone substrates with different substituents at phenyl group was investigated. The results reveal that the para -position of diaryl ketones plays important role in manipulating the stereoselectivity of Kp ADH. Two vital residues, E214 and T215, were identified through single-point mutagenesis, and saturation mutagenesis was performed to evaluate their contributions to the stereoselectivity. Furthermore, stereodivergent evolution of Kp ADH was achieved through combinatorial mutagenesis. Among them, E214I/T215S/S237A (ISA) and F161V/S196G/E214G (VGG) exhibited e.e. values of >99% ( S ) and 99% ( R ) toward [4-(trifluoromethyl)phenyl]-2-pyridinylmethanone ( 5a ), and e.e. values of >99% ( S ) and 98% ( R ) toward (4-methylphenyl)-2-pyridinylmethanone ( 6a ), respectively. Kinetic characterization and interaction analysis further prove that the eliminated side-chain collision and enhanced electrostatic interactions are mainly responsible for the increased catalytic efficiency and stereoselectivity. This study provides beneficial mutants with complementary stereoselectivity toward diaryl ketones with para -substituents and also provides guidance for the engineering of homologous alcohol dehydrogenases toward bulky-bulky ketones.
研究领域:
生物化学
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酶工程
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DOI
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2024
·Vol. 91
·期 1
·
IF 3.9
Yuan-Ru Xiong,Yuan-Chun Fang,Min He,Ke-Jing Li,Lei Qi,Yang Sui,Ke Zhang,Xue-Chang Wu,Liang Meng,Ou Li,Dao-Qiong Zheng
This study explored the genomic alterations in Yarrowia lipolytica, a key yeast in industrial biotechnology, under both spontaneous and mutagen-induced conditions. Our findings reveal that spontaneous mutations occur at a rate of approximately 4 × 10−10 events per base pair per cell division, primarily manifesting as single-nucleotide variations (SNVs) and small insertions and deletions (InDels). Notably, C-to-T/G-to-A transitions and C-to-A/G-to-T transversions dominate the spontaneous SNVs, while 1 bp deletions, likely resulting from template slippage, are the most frequent InDels. Furthermore, chromosomal aneuploidy and rearrangements occur, albeit at a lower frequency. Exposure to ultraviolet (UV) light, methylmethane sulfonate (MMS), and Zeocin significantly enhances the rates of SNVs and alters their mutational spectra in distinct patterns. Notably, Zeocin-induced SNVs are predominantly T-to-A and T-to-G substitutions, often occurring within the 5′-TGT*−3′ motif (* denotes the mutated base). Additionally, Zeocin exhibits a higher potency in stimulating InDels compared to UV and MMS. Translesion DNA synthesis is implicated as the primary mechanism behind most Zeocin-induced SNVs and some InDels, whereas non-homologous end joining serves as the main pathway for Zeocin-mediated InDels. Intriguingly, the study identifies the gene YALI1_E21053g, encoding a protein kinase, as negatively associated with Zeocin resistance. Overall, our results not only deepened our knowledge about the genome evolution in Y. lipolytica but also provided reference to develop innovative strategies to harness its genetic potential.
研究领域:
微生物学
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遗传学
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分子生物学
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DOI
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2024
·
IF 7.7
Lingzhi Zhang,Rui Zhou,Dekai Liu,Meinan Zhu,Guangya Zhang,Lijuan Zhang,Shu-Feng Zhou,Wei Jiang
Given their outstanding efficiency and selectivity, enzymes are integral in various domains such as drug synthesis, the food industry, and environmental management. However, the inherent instability of natural enzymes limits their widespread industrial application. In this study, we underscore the efficacy of enhancing protein thermal stability through comprehensive protein design strategies, encompassing elements such as the free energy of protein folding, internal forces within proteins, and the overall structural design. We also demonstrate the efficiency and precision of combinatorial screening in the thermal stability design of aldo-keto reductase (AKR7-2-1). In our research, three single-point mutations and five combinatorial mutations were strategically introduced into AKR7-2-1, using multiple computational techniques. Notably, the E12I/S235I mutant showed a significant increase of 25.4 °C in its melting temperature ( Tm ). Furthermore, the optimal mutant, E12V/S235I, maintained 80 % of its activity while realizing a 16.8 °C elevation in Tm . Remarkably, its half-life at 50 °C was increased to twenty times that of the wild type. Structural analysis indicates that this enhanced thermal stability primarily arises from reduced oscillation in the loop region and increased internal hydrogen bonding. The promising results achieved with AKR7-2-1 demonstrate that our strategy could serve as a valuable reference for enhancing the thermal stability of other industrial enzymes.
研究领域:
生物化学
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分子生物学
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工业酶工程
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DOI
PubMed
高影响因子
Experimental and Therapeutic Medicine
·
2019
·Vol. 18
·期 4
·3117-3124
·
IF 1.448
Qiurong Zhang,Xiao Wu,Jing Cao,Feng Gao,Kun Huang
A total of 133 patients with acute myeloid leukemia (AML) were enrolled in the current study and were subdivided into 4 groups: 34 harboring DNA methyltransferase 3 α (DNMT3A) + fms related tyrosine kinase 3‑internal tandem duplication (FLT3‑ITD) mutations, 37 harboring only FLT3‑ITD mutation, 32 harboring only DNMT3A mutation and 30 harboring no mutations in DNMT3A and FLT3‑ITD (control). Patients in all groups were administered daunorubicin and cytarabine chemotherapy regimens. The rates of complete remission (CR), 1‑year relapse (RR) and 3‑year overall survival (OS) were compared. Patients in the DNMT3A + FLT3‑ITD mutation group exhibited higher proportions of peripheral white blood cells (WBCs) and myeloid progenitor cells compared with those in DNMT3A mutation only, FLT3‑ITD mutation only and control groups (P<0.05). The rates of CD15+ and HLA‑DR+ in the DNMT3A + FLT3‑ITD mutation and DNMT3A mutation only groups were significantly higher than those in the FLT3‑ITD mutation only and control groups (P<0.05); in addition, the rate of CD38+ in the DNMT3A + FLT3‑ITD mutation and FLT3‑ITD mutation only groups was significantly higher compared with that in the DNMT3A mutation only and control groups (P<0.05). The overall chemotherapy effectiveness rate, CR, 1‑year RR and the 3‑year OS rates of patients in the DNMT3A + FLT3‑ITD mutation group were significantly worse compared with FLT3‑ITD mutation only, DNMT3A mutation only and control groups (P<0.05). The results of this study indicated that increased mutation rates in DNMT3Α and FLT3‑ITD may be associated with increased WBC and myeloid progenitor cell counts, an inferior chemotherapy efficacy and prognosis, a lower CR rate, and higher 1‑year RR and mortality rate.
研究领域:
血液学
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肿瘤学
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分子生物学
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产品关联
DOI
PubMed
高影响因子
MOLECULES
·
2024
·Vol. 29
·期 19
·4713
·
IF 4.2
Wei He,Qi Geng,Guiying Ji,Ji Li,Dan Wang,Yucai He,Qiuheng Jin,Jianren Ye
The remarkable efficacy of COVID-19 vaccines has established mRNA as a highly promising biomedical technology. However, the adequate application of mRNA therapeutics necessitates additional measures to mitigate the inherent immunogenicity, which is predominantly caused by dsRNA. As a byproduct of the in vitro transcription of mRNA, dsRNA was reported to be originated through several distinct mechanisms, including the extension of 3′ loop-back hairpins, the extension of hybridized abortive transcripts, and promoter-independent transcription. The intricate mechanisms involved pose a dilemma as the reduction in dsRNA results in a concomitant decrease in other critical quality attributes of mRNA. Here, we demonstrate that the promoter binding motifs of T7 RNA polymerase directly impact the production of promoter-independent transcription-based dsRNA. Specifically, the G753A mutation significantly reduces the formation of dsRNA byproducts, which can further combine with modified nucleotides to enhance the effectiveness of dsRNA mitigation and with previously reported high-integrity mutation K389A to minimize side effects. Accordingly, the present study reports a cost-effective approach to synthesize high-purity, less immunostimulatory mRNA by using an engineered T7 RNA polymerase mutant.
研究领域:
分子生物学
·
生物技术
·
药学
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产品关联
DOI
PubMed
高影响因子
Foods
·
2024
·Vol. 13
·期 9
·1404
·
IF 4.7
Yuyang Yao,Juer Liu,Qiming Miao,Xinyue Zhu,Wei Hua,Na Zhang,Guangwei Huang,Xiangyang Lin,Shengquan Mi,Yanling Cheng,Roger Ruan
Almond hull, a substantial byproduct comprising more than half of almond fresh weight, has recently gained attention due to its functionality and sustainability benefits. Despite heightened interest, information regarding its toxicity remains limited. In order to assess its genotoxic potential, we conducted Good Laboratory Practice-compliant in vitro and in vivo studies following Organization for Economic Co-operation and Development (OECD) guidelines. No evidence of toxicity or mutagenicity was observed in a bacterial reverse mutation assay using five tester strains, evaluating almond hull at concentrations up to 5 mg/plate, with or without metabolic activation. Almond hull did not induce chromosome structural damage in a chromosome aberration assay using Chinese hamster ovary cells, nor did it cause any spermatogonial chromosomal aberration in tested male BALB/c mice. To evaluate its ability to induce DNA damage in rodents, a combined micronucleus assay was conducted in KM mice of both sexes. Almond hull was administered at doses of 1250, 2500, and 5000 mg/kg/day via gavage once daily for 2 days. No adverse effects of almond hull were observed in the micronucleus assay. Our results indicate no evidence of the genotoxic potential of almond hull administered up to the maximum concentrations of 5 g/kg, as recommended by OECD guidelines.
研究领域:
食品科学
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毒理学
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遗传学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
iScience
·
2024
·
IF 4.6
Jiawei Wang,Xingyu Ouyang,Shiyu Meng,Jiayi Li,Liangxu Liu,Chaofeng Li,Hengrun Li,Haotian Zheng,Chao Liao,Yi-Lei Zhao,Jun Ni
Lignin valorization is crucial for achieving economic and sustainable biorefinery processes. However, the enzyme substrate preferences involved in lignin degradation remain poorly understood, and low activity toward specific substrates presents a significant challenge to the efficient utilization of lignin. In this study, we investigated the substrate promiscuity of Th Ado, a key enzyme involved in lignin valorization. Pre-reaction state analysis revealed that a hydrogen bond network is critical in determining substrate selectivity. By performing targeted saturation mutagenesis on residues surrounding the substrate tunnels, we identified the Y205W and Y205Q mutants, which demonstrated 0.73-fold and 0.72-fold enhancements in activity, respectively. Structural analysis indicated that the redirection of the original substrate tunnel may be responsible for the improved activity. Our study provides essential insights into the substrate preference mechanisms of lignin degrading enzymes and suggests that this tunnel-redirection strategy can be extended to other promiscuous enzymes.
研究领域:
生物化学
·
生物技术
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酶学
·
可持续化学
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产品关联
DOI
PubMed
高影响因子
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
·
2022
·Vol. 88
·期 9
·
IF 5.005
Dong-Xu Jia,Fan Wang,Rui Zhao,Bo-Di Gu,Chen Peng,Li-Qun Jin,Zhi-Qiang Liu,Yu-Guo Zheng
d-Alanine belongs to nonessential amino acids that have diverse applications in the fields of food and health care. (R)-transaminase [(R)-TA]-catalyzed asymmetric amination of pyruvate is a feasible alternative for the synthesis of d-alanine, but low catalytic efficiency and thermostability limit enzymatic utilization. In this work, several potential (R)-TAs were discovered using NCBI database mining synchronously with enzymatic structure-function analysis, among which Capronia epimyces TA (CeTA) showed the highest activity for amination of pyruvate using (R)-α-methylbenzylamine as the donor. Furthermore, enzymatic residues surrounding a large catalysis pocket were subjected to saturation and combinatorial mutagenesis, and positive mutant F113T showed dramatic improvement in activity and thermostability. Molecular modeling indicated that the substitution of phenylalanine with threonine afforded alleviation of steric hindrance in the pocket and induced formation of additional hydrogen bonds with neighboring residues. Finally, using recombinant cells containing F113T as a biocatalyst, the conversion yield of amination of 100 mM pyruvate to d-alanine achieved up to 95.2%, which seemed to be the highest level in the literature regarding synthesis of d-alanine using TAs. The inherent characteristics rendered CeTA F113T a promising platform for efficient preparation of d-alanine operating with high productivity.IMPORTANCE d-Alanine is an important compound with many valuable applications. Its asymmetric synthesis employing (R)-ω-TA is considered an attractive choice. According to the stereoselectivity, ω-TAs have either (R)- or (S)-enantiopreference. There has been a variety of literature regarding screening, characterizing, and molecular modification of (S)-ω-TAs; in contrast, the research about (R)-ω-TA has lagged behind. In this work, we identify several (R)-ω-TAs and succeeded in creating mutant F113T, which showed not only better efficiency toward pyruvate but also higher thermostability compared with the original enzyme. The obtained original enzymes and positive mutants displayed important application value for pushing symmetric synthesis of d-alanine to a higher level.
研究领域:
生物技术
·
酶工程
·
化学生物学
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产品关联
DOI
PubMed
高影响因子
ANALYTICAL CHEMISTRY
·
2020
·
IF 6.785
Jing Lei,Yuqin Huang,Wenjun Zhong,Dan Xiao,Cuisong Zhou
With the aim of detecting low frequency of drug resistant mutation T790M against wild-type sequences, we reported a two-dimensional signal analysis strategy by combining a three locked nucleic acids (LNAs)-modified probe (LP15-3t) and an α-HL nanopore. The specific hybridization of the LP15-3t probe with the T790M generated unique long two-level signals, including characteristic blocking current and characteristic dwell time. Due to the significant dwell time difference (114.2-fold) and the blocking current difference ranging from 81% to 96%, this two-dimensional signal analysis strategy can simultaneously distinguish T790M sequences with a sensitivity of 0.0001% against wild-type sequences. The LOD of T790M was 0.1 pM. This high discrimination capability would have great potential in the detection of rare mutation sequences and the early monitoring of clinical outcome of NSCLC patients with TKI drug resistance.
研究领域:
分子生物学
·
医学诊断
·
纳米科技
✓ 已验证
DOI
高影响因子
APPLIED MICROBIOLOGY AND BIOTECHNOLOGY
·
2017
·Vol. 101
·期 13
·5325-5332
·
IF 3.42
Cao Hao,Nie Kaili,Li Chengcheng,Xu Haijun,Wang Fang,Tan Tianwei,Liu Luo
Adenosine-5′-triphosphate (ATP) is the energy equivalent of the living system. Polyphosphate (polyP) is the ancient energy storage equivalent of organisms. Polyphosphate kinases (PPKs) catalyze the polyP formation or ATP formation, to store energy or to regenerate ATP, respectively. However, most PPKs are active only in the presence of long polyPs, which are more difficult and more expensive to generate than the short polyPs. We investigated the PPK preference towards polyPs by site-directed mutagenesis and computational simulation, to understand the mechanism and further design enzymes for effective ATP regeneration using short polyPs for in vitro cascade reactions, which are highly desired for research and applications. The results suggest that the short polyPs inhibit PPK by blocking the ADP-binding pocket. Structural comparison between PPK ( Corynebacterium glutamicum ) and PPK ( Sinorhizobium meliloti ) indicates that three amino acid residues, i.e., lysine, glutamate, and threonine, are involved in the activity towards short polyP by fixing the adenosine group of ADP in between the subunits of the dimer, while the terminal phosphate group of ADP still offers an active site, which presents a binding pocket for ADP. A proposed triple mutant PPK (SMc02148-KET) demonstrates significant activity towards short polyP to form ATP from ADP. The obtained high glutathione titer (38.79 mM) and glucose-6-phosphate titer (87.35 mM) in cascade reactions with ATP regeneration using the triple mutant PPK (SMc02148-KET) reveal that the tailored PPK establishes the effective ATP regeneration system for ATP-dependent reactions.
研究领域:
生物化学
·
酶工程
·
合成生物学
✓ 已验证
DOI
PubMed
高影响因子
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