Redox Biology
·
2024
·
IF 10.7
Wuyang Shi,Shibo Sun,Haowen Liu,Yao Meng,Kangshuai Ren,Guoying Wang,Minghui Liu,Jiaqi Wu,Yue Zhang,Huang Huang,Meiyun Shi,Weiping Xu,Qiang Ma,Bingbing Sun,Jianqiang Xu
Thioredoxin reductase (TXNRD) is a selenoprotein that plays a crucial role in cellular antioxidant defense. Previously, a distinctive guiding bar motif was identified in TXNRD1, which influences the transfer of electrons. In this study, utilizing single amino acid substitution and Excitation-Emission Matrix (EEM) fluorescence spectrum analysis, we discovered that the guiding bar communicates with the FAD and modulates the electron flow of the enzyme. Differential Scanning Fluorimetry (DSF) analysis demonstrated that the aromatic amino acid in guiding bar is a stabilizer for TXNRD1. Kinetic analysis revealed that the guiding bar is vital for the disulfide reductase activity but hinders the selenocysteine-independent reduction activity of TXNRD1. Meanwhile, the guiding bar shields the selenocysteine residue of TXNRD1 from the attack of electrophilic reagents. We also found that the inhibition of TXNRD1 by caveolin-1 scaffolding domain (CSD) peptides and compound LCS3 did not bind to the guiding bar motif. In summary, the obtained results highlight new aspects of the guiding bar that restrict the flexibility of the C-terminal redox motif and govern the transition from antioxidant to pro-oxidant.
✓ 已验证
产品关联
DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
·
2020
·
IF 4.192
Yidong Zhou,Zhijian Ke,Hangting Ye,Mengting Hong,Yifei Xu,Mingliang Zhang,Wankui Jiang,Qing Hong
Carbaryl is a widely used carbamate pesticide in agriculture. The strain Rhizobium sp. X9 possesses the typical carbaryl degradation pathway in which carbaryl is mineralized via 1-naphthol, salicylate, and gentisate. In this study, we cloned a carbaryl hydrolase gene cehA and a novel two-component 1-naphthol hydroxylase gene cehC1C2. CehA mediates carbaryl hydrolysis to 1-naphthol and CehC1, an FMNH2 or FADH2-dependent monooxygenase belonging to the HpaB superfamily, and hydroxylates 1-naphthol in the presence of reduced nicotinamide-adenine dinucleotide (FMN)/flavin adenine dinucleotide (FAD), and the reductase CehC2. CehC1 has the highest amino acid similarity (58%) with the oxygenase component of a two-component 4-nitrophenol 2-monooxygenase, while CehC2 has the highest amino acid similarity (46%) with its reductase component. CehC1C2 could utilize both FAD and FMN as the cofactor during the hydroxylation, although higher catalytic activity was observed with FAD as the cofactor. The optimal molar ratio of CehC1 to CehC2 was 2:1. The Km and Kcat/Km values of CehC1 for 1-naphthol were 74.71 ± 16.07 μM and (8.29 ± 2.44) × 10–4 s–1·μM–1, respectively. Moreover, the enzyme activities and substrate spectrum between CehC1C2 and previously reported 1-naphthol hydroxylase McbC were compared. The results suggested that McbC had a higher 1-naphthol hydroxylation activity, while CehC1C2 had a broader substrate spectrum.
研究领域:
环境微生物学
·
生物化学
·
农药生物降解
✓ 已验证
产品关联
DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
·
2018
·
IF 3.909
Cen Zhang,Rui Wang,Guowen Zhang,Deming Gong
Quercetin, one of the most abundant flavonoid in the daily diet, was found to reversibly inhibit the generation of uric acid and superoxide radicals (O 2 − )catalyzed by xanthine oxidase (XOD) in a mixed-type manner with IC 50 values of (2.74 ± 0.04) × 10 −6 and (2.90 ± 0.03) × 10 −6 mol L −1 , respectively, and the inhibition of quercetin on O 2 − generation may be ascribed to the reduced form of XOD by a ping-pong mechanism. XOD had one high affinity binding site for quercetin with a binding constant of 4.28 × 10 4 L mol −1 at 298 K, and the binding process was predominately driven by van der Waals forces and hydrogen bonds on account of the negative enthalpy and entropy changes. Moreover, molecular docking confirmed that the binding site for quercetin located in the isoalloxazine ring of the flavin adenine dinucleotide (FAD) domain of XOD, then the diffusion of O 2 − out of the FAD site was blocked in favor of another electron transferred from FADH 2 to O 2 − to form hydrogen peroxide (H 2 O 2 ). This study may clarify the role of quercetin on inhibiting XOD catalysis and provide a potential nutritional supplement for preventing gout and peroxidative damage.
研究领域:
生物化学
·
药理学
·
营养科学
✓ 已验证
产品关联
DOI
PubMed
高影响因子
ACS Sustainable Chemistry & Engineering
·
2019
·
IF 6.97
Yan Zhang,Yin-Qi Wu,Na Xu,Qian Zhao,Hui-Lei Yu,Jian-He Xu
Enzymatic asymmetric sulfoxidation using molecular oxygen as the oxidant is a promising green chemistry approach to chiral sulfoxide production. Despite the broad substrate spectrum of cyclohexanone monooxygenases (CHMOs), some unnatural substrates with bulky functional groups, such as the pharmaceutically relevant omeprazole sulfide, cannot be effectively accepted by CHMOs. Herein, we describe a set of variants derived from an Acinetobacter calcoaceticus CHMO (AcCHMO), whose active sites adjacent to the substrate tunnel were altered to shift the substrate specificity from cyclohexanone monooxygenation toward omeprazole sulfide sulfoxidation. We performed homologous modeling and molecular docking to identify key residues that might affect the substrate specificity. Two libraries of residues lining the active center of AcCHMO were then constructed and screened by an effective halo-based selection method using the solubility difference between the substrate (omeprazole sulfide) and product (esomeprazole). Functional evaluation of the resultant variants showed that the substrate specificity of AcCHMO was markedly altered from the small natural substrate (cyclohexanone) toward the desired bulky substrate (omeprazole sulfide) despite the extremely poor activity detected even for the best variant, M2 (0.61 U/gprot). The crystal structure of M2 complexed with a flavin adenine dinucleotide (FAD) prosthetic group was determined, which provided insight into the altered substrate specificity. To improve the activity of enzyme M2 toward pharmaceutical precursor omeprazole sulfide, we performed both local and global protein engineering among the two CASTing libraries surrounding FAD+ and NADP+ prosthetic groups and an error-prone PCR library of the full-length AcCHMO. As a result, variant M6 was obtained, giving a 50-fold higher activity compared to M2. This structure-guided protein engineering of AcCHMO provided a promising candidate for converting omeprazole sulfide into (S)-omeprazole using a green biocatalytic method.
研究领域:
生物催化
·
酶工程
·
药物化学
·
绿色化学
·
蛋白质结构与功能
✓ 已验证
DOI
高影响因子
危险品化学品经营许可证（带存储）