Frontiers in Microbiology
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2021
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IF 5.64
Liu Xinning,Liu Yuanchang,Zhao Guangjian,Zhang Yidan,Liu Lu,Wang Juan,Wang Yifan,Zhang Siyu,Li Xin,Guo Dongliang,Wang Peng,Xu Ximing
Vibrio vulnificus is a zoonotic bacterium that is capable of causing highly lethal diseases in humans; this pathogen is responsible for 95% of all seafood-related deaths in the United States. Arylamine N-acetyltransferases (NAT, E.C. 2.3.1.5) is a major family of xenobiotic-metabolizing enzymes that can biotransform aromatic amine chemicals. In this research, to evaluate the effect of NAT on acetyl group transformation in arylamine antibiotics, we first used sequence alignment to study the structure of V. vulnificus NAT [(VIBVN)NAT]. The nat gene encodes a protein of 260 amino acids, which has an approximate molecular mass of 30 kDa. Then we purified recombinant (VIBVN)NAT and determined the enzyme activity by PNPA and DTNB methods. The DTNB method indicates that this prokaryotic NAT has a particular substrate specificity towards aromatic substrates. However, (VIBVN)NAT lost most of its activity after treatment with high concentrations of urea and H2O2. In addition, we also explored the stability of the enzyme at different temperatures and pH values. In analyzing the influence of metal ions, the enzyme activity was significantly inhibited by Zn2+ and Cu2+. The kinetic parameters Km and Vmax were determined using hydralazine, isoniazid, 4-amino salicylic acid, and 4-chloro-3-methylaniline as substrates, and the Tm, Tagg and size distribution of (VIBVN)NAT were observed. In particular, a molecular docking study on the structure of (VIBVN)NAT was conducted to understand its biochemical traits. These results showed that (VIBVN)NAT could acetylate various aromatic amine substrates and contribute to arylamine antibiotic resistance in V. vulnificus.
研究领域:
微生物学
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生物化学
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分子生物学
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药理学
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PubMed
高影响因子
Forensic Chemistry
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2023
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IF 2.7
Jiaxin Wang,Weimin Wang,Di Zhang,Fangling Wu,Chuan-Fan Ding
In this study, a simple and accurate method for the separation and quantitative analysis of cinchona alkaloid stereoisomers and analogues was developed using trapped ion mobility spectrometry (TIMS)-mass spectrometry (MS) and theoretical calculations. Stereoisomer studied were N-(9-Deoxy- epi -cinchonin-9-yl) picolinamide ( N -CNP)/N-(9-Deoxy- epi -cinchonidin-9-yl) picolinamide ( N -CDP), N -Benzylcinchoninium (BCN)/ N -Benzylcinchonidinium (BCN), and Cinchonine (CCN)/Cinchonidine (CCD). TIMS results revealed slight mobility differences in the protonated stereoisomers, [CCD + H] + /[CCN + H] + complexes showed the most effective separation, with a resolution (R P–P ) of 0.431. When the macrocyclic antibiotics rifamycin (Rif) and natamycin (Nat) were introduced as separation reagents, the binary diastereomer complexes [Nat/Rif + CCD/CCN + H] + , [Rif + N -CDP/ N -CNP + H] + , and [Rif + BCD/BCN + H] + were formed by non-covalent interaction with the stereoisomers. The TIMS analysis revealed that the stereoisomers were separated by the formed binary complexes: CCD/CCD was separated by [CCD/CCN + Nat + H] + (R P–P = 1.771), N -CDP/ N -CNP was separated by [ N -CDP/ N -CNP + Rif + H] + (R P–P = 0.914), and the BCD/BCN was separated by [BCD/BCN + Rif] + (R P–P = 0.713). The conformations of the binary complexes were optimized using chemical theoretical calculations which revealed the microscopic interaction between the stereoisomers was different, and that the theoretical collision cross section (CCS) was consistent with the observed experimental results. An additional quantitative analysis of the stereoisomers yielded a limit of detection (LOD) ≦ 0.408 μg L −1 and satisfactory linearities (R 2 > 0.99). The proposed method is simple, highly selective, highly sensitive, and without the need for chromatographic separation, and is therefore a promising option for the separation and analysis of cinchonine stereoisomers or analogues, as well as in desirable figures-of-merit for forensics.
研究领域:
分析化学
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药学
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生物信息学
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DOI
高影响因子
ANALYTICA CHIMICA ACTA
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2022
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IF 6.911
Yiyi Liu,Fangling Wu,Jiabin Wang,Ling Pu,Chuan-Fan Ding
The separation of chiral amino acids (AAs) and their derivatives has always been a research difficulty in the field of biochemistry due to the high similarity of enantiomeric structures. In this work, a simple and quick method using natamycin (Nat) as chiral selector has been developed to simultaneously separate chiral AAs and their derivatives of carbobenzoxy/benzyl-AAs (Cbz/Bzl-AAs) by trapped ion mobility spectrometry-mass spectrometry (TIMS-MS). Specifically, 12 groups of the Cbz-AAs and Bzl-AAs can get baseline mobility separation by simple mixing with Nat to form binary diastereomeric complex ions [Nat+(Cbz-D/L-AA)+H] + and [Nat+(Bzl-D/L-AA)+H] + . While for the remained 5 groups of Bzl-D/L-AAs and 16 groups of D/L-AAs with unsatisfying separation, by further adding P-toluenesulfonic acid (PTS), the formed ternary complexes can allow their baseline chiral separation . Specifically, Bzl-D-AAs and Bzl-L-AAs get much improved separation effect by the formed diastereomeric complexes of [Nat+(Bzl-AA) 2 +PTS 2 +H] + , which the R p-p was improved from 0 to 2.40; while the D/L-AAs can get baseline separation by the formed diastereomeric complexes of [Nat + AA + PTS + H] + , [Nat + AA+(PTS) 2 +H] + , and [Nat+(AA) 2 +(PTS) 2 +H] + , with the R p-p ranged from 0.44 to 3.53. Definitely, PTS is the first time reported as the ligand to improve the separation effect for the enantiomers, and with the higher assembly of chiral analyte, Nat, and PTS might enable better chiral separation for the chiral amino acid and their derivatives. Moreover, method validation of relative quantification and accuracy for the D/L-AA and their derivatives were measured in a mixture, yielding R 2 greater than 0.99 and RSD% ≤ 2.68%. Overall, Nat and PTS as chiral selector and ligand can be widely used for chiral AAs and their derivatives mobility separation, and potentially for the separation of other AA-related chiral molecules.
研究领域:
生物化学
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分析化学
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DOI
PubMed
高影响因子
MICROCHEMICAL JOURNAL
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2023
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IF 4.8
Sisi He,Yiyi Liu,Chaoxian Chi,Dongdong Zhou,Chuan-Fan Ding,Xucong Lin,Jiabin Wang,Fangling Wu
The differentiation of 3-AminoPyrrolidine (3AP) and its derivative isomers is significant due to their use as intermediates in drug synthesis. However, the separation of these isomers, which exhibit both chiral and positional isomerism, has consistently posed challenges. In this study, an efficient analytical method was developed to discrimination between the four isomers of R/S-I/N-Boc-3-AminoPyrrolidine (R/S-l/N-Boc-3AP) and four groups of derived chiral isomers, namely R/S-N-Methyl-3-AminoPyrrolidine (R/S-N-Me-3AP), R/S-N-Ethyl-3-AminoPyrrolidine (R/S-N-Et-3AP), R/S-1-Bzl-3-AminoPyrrolidine (R/S-1-Bzl-3AP), and R/S-1-Cbz-3-AminoPyrrolidine (R/S-1-Cbz-3AP). The discrimination was achieved by utilizing natamycin (Nat) as a differentiation agent and 2/3/4-aminobenzenesulphonic acid (2/3/4-ABSA) as ligand. The facile discrimination of the four positional isomers of R/S-1-Boc-3AP and R/S-N-Boc-3AP was achieved using ion mobility spectrometry (IMS) based on their formed diastereomeric complexes [Nat + (Boc-3AP) 2 + (2-ABSA) 2 + H] + . The isomers were also studied via tandem mass spectrometry (MS/MS) using the formed ternary complexes, different fragment ions were observed for l-Boc-3AP and N-Boc-3AP, but the R/S enantiomers cannot be discriminated either. In addition, the enantiomers R/S-l-Bzl-3AP and R/S-l-Cbz-3AP were differentiated based on mobility using [Nat + (R/S-l-Bzl-3AP) + (3-ABSA) 2 + H] + and [Nat + (R/S-l-Cbz-3AP) + 2-ABSA + H] + ; however for the small enantiomeric molecules of R/S-N-Me-3AP and R/S-N-Et-3AP, chiral discrimination was not achieved via the ternary complexes of Nat + R/S-N-Me-3AP-ABSA and Nat + R/S-N-Et-3AP-ABSA. Importantly, the separation was greatly improved by the coordinated alkali metal ions, where the separation resolution (R p-p ) increased with the radius of the metal ions. Finally, quantitative analysis and method validation were carried out for the four Boc-3AP isomers using both MS/MS and IMS-MS. Overall, the proposed method opens possibilities for the rapid discrimination and quantification of isomers exhibiting both chiral and positional isomerism in MS/MS and IMS-MS analyses.
研究领域:
分析化学
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药物化学
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质谱分析
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DOI
高影响因子
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