ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS
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2024
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IF 3.8
Yan-Fei Liang,Ze-Xin Niu,Zi-Wen Wu,Qing-Yang Zhang,Xin-Yi Zhao,Lei-Lei Chao,Heng Li,Wen-Yun Gao
Acetolactate synthase (ALS) is an essential enzyme involved in the biosynthesis of platform chemicals acetoin and 2,3-butanediol in several microorganisms. In this study, we investigated the catalytic differences among three bacterial ALSs involved in the ligation of two molecules of pyruvate or 2-ketobutyrate. Based on the findings, we predicted three amino acid residues in each enzyme that caused a discrepancy in accordance with the multi-sequence alignment and molecular docking experiments: I398, A402, and T480 in Bacillus subtilis ALS; V400, Y404, and S482 in Listeria seleigeri serovar 1/2b ALS; and M394, H398, and G476 in Klebsiella pneumoniae ALS. Subsequently, we mutually mutated the residues in the three ALSs. The data obtained confirmed our inference that these three residues in each enzyme are truly correlated with substrate recognition, particularly in recognizing compounds that are larger than pyruvate, such as 2-ketobutyrate, benzaldehyde, and nitrosobenzene. This study further clarifies the biochemical traits of ALSs derived from various bacteria and expands the scope of ALS research.
研究领域:
生物化学
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微生物学
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DOI
PubMed
高影响因子
CARBOHYDRATE POLYMERS
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2024
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IF 10.7
Xueyan Wang,Le Liu,Ruiyu Shen,Qian Wang,Xiaoqi Xie,Weiming Liu,Zhimin Yu,Xianzhen Li,Xiaoyu Guo,Fan Yang
The inefficient decomposition of polysaccharides, particularly branched polysaccharides limits their large-scale industrial applications. Further understanding and modification of glycoside hydrolases (GHs) processivity is expected to overcome this limitation. Here, a novel xanthan-binding CBM ( Mi XBM), which was supposed to alter the processivity of GHs, was systematically characterized. Phylogeny and structure analyses indicated that Mi XBM is closely related to putative polysaccharide side chain-binding modules. Quantitative binding assays further revealed that Mi XBM probably has a high affinity for xanthan side chain via a variable loop site. Moreover, catalytic performance demonstrated that xanthanase chimeras containing Mi XBM promote highly efficient hydrolysis of xanthan because of improved substrate accessibility. Notably, Mi XBM was observed to enhance the processivity of xanthanase, owing to its high substrate affinity to the repeating unit xanthan. Furthermore, sequential hydrolysis of xanthan by xanthanases with varying processivity resulted in significantly increased hydrolytic efficiency and focused oligoxanthans array. These results expand understanding of CBM-substrate recognition and shed light on efficient degradation of other regularly branched polysaccharides using modified GHs.
CAS / MDL:
研究领域:
生物化学
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分子生物学
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DOI
PubMed
高影响因子
ANALYTICAL LETTERS
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2025
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IF 1.8
Lucheng Chen,Jiaying Yu,Qi Liu,Jiaxing Han,Daoxiang Su,Zhifei Wang
Carboxylesterase 2 (CES2) plays critical roles in lipid metabolism and exogenous detoxification, yet current detection methods lack specificity in complex biological systems. To overcome this limitation, we developed CLC-CES2, a near-infrared fluorescent probe rationally designed based on CES2's substrate recognition mechanisms. This probe demonstrates exceptional performance with near-infrared emission (695 nm), a large Stokes shift (145 nm), and a low detection limit (0.021 μg/mL). Biological validation using live cell imaging and zebrafish models demonstrated successful detection of endogenous CES2 activity. These results establish CLC-CES2 as an efficient molecular tool for investigating CES2- associated physiological and pathological processes.
研究领域:
生物化学
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分子生物学
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医学影像学
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DOI
高影响因子
IUBMB LIFE
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2018
·Vol. 71
·期 3
·330-339
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IF 3.236
Qing Wang,Maikun Teng,Xu Li
Catechol- O -methyltransferase (COMT 1 ) catalyzes the transfer of a methyl group from S-adenosylmethionine (SAM) to various catechol substrates. COMTs play vital roles in physiological processes in animals, plants, and fungi, as well as bacteria, and have essential application values in industry. sp COMT is a probable COMT from Schizosaccharomyces pombe . It has an extraordinary intracellular distribution different from other homologs and would thus be predicted to perform a distinct physiological function. In this report, recombinant sp COMT was purified and kinetically characterized for the first time. The enzymology assays indicate that sp COMT is a metal-dependent enzyme and belongs to class I OMTs. In addition, the crystal structures of apo- sp COMT and SAM-complexed sp COMT were also presented, revealing that sp COMT possesses a conserved SAM-binding site and Mg 2+ pocket, but a distinct substrate pocket was not present in homologs. The mutagenesis ITC analysis revealed the SAM recognition characteristics of sp COMT. Based on all of the above findings, we speculated about the putative substrates’ characteristics and the substrate recognition mechanisms of sp COMT. This work will help in elucidating the physiological functions of sp COMT in S. pombe . © 2018 IUBMB Life, 71(3):330–339, 2019
研究领域:
生物化学
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分子生物学
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结构生物学
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DOI
PubMed
高影响因子
CELL RESEARCH
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2024
·Vol. 34
·期 1
·47-57
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IF 28.1
Wang Yuwei,Zhang Pei,Chao Yulin,Zhu Zhini,Yang Chuanhui,Zhou Zixuan,Li Yaohui,Long Yonghui,Liu Yuehua,Li Dianfan,Wang Sheng,Qu Qianhui
Monoamine neurotransmitters such as serotonin and dopamine are loaded by vesicular monoamine transporter 2 (VMAT2) into synaptic vesicles for storage and subsequent release in neurons. Impaired VMAT2 function underlies various neuropsychiatric diseases. VMAT2 inhibitors reserpine and tetrabenazine are used to treat hypertension, movement disorders associated with Huntington’s Disease and Tardive Dyskinesia. Despite its physiological and pharmacological significance, the structural basis underlying VMAT2 substrate recognition and its inhibition by various inhibitors remains unknown. Here we present cryo-EM structures of human apo VMAT2 in addition to states bound to serotonin, tetrabenazine, and reserpine. These structures collectively capture three states, namely the lumen-facing, occluded, and cytosol-facing conformations. Notably, tetrabenazine induces a substantial rearrangement of TM2 and TM7, extending beyond the typical rocker-switch movement. These functionally dynamic snapshots, complemented by biochemical analysis, unveil the essential components responsible for ligand recognition, elucidate the proton-driven exchange cycle, and provide a framework to design improved pharmaceutics targeting VMAT2.
研究领域:
神经科学
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药理学
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结构生物学
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DOI
PubMed
高影响因子
JOURNAL OF AGRICULTURAL AND FOOD CHEMISTRY
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2025
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IF 6.2
Yanbin Feng,Nuanqing Wang,Qianqian Cao,Shang Li,Ying Xu,Xinyu Che,Jinfeng Zhao,Chuanlun Yang,Song Xue
Trehalose 6-phosphate (Tre6P) is a critical metabolic signaling molecule in plants, orchestrating diverse biological processes, including stress resistance and photosynthetic efficiency. Recent advancements highlight its promising role in improving crop yield. However, the efficient synthesis of Tre6P in vitro remains a major challenge. Here, we identified a trehalose-6-phosphate phosphorylase WcTre6PPase from Weissella ceti, the enzyme catalyzes the reversible synthesis of Tre6P from β-glucose 1-phosphate (βGlc1P) and glucose 6-phosphate (Glc6P), with a strong catalytic bias for Tre6P synthesis. The crystal structure of WcTre6PPase resolved in complex with βGlc1P reveals critical molecular determinants for substrate recognition and catalytic efficiency. Binding studies demonstrate a higher affinity of WcTre6PPase for βGlc1P by 1.0 ± 0.2 mM compared to Glc6P by 24.3 ± 7.1 mM. Complementary molecular docking and dynamics simulations provide detailed insights into the catalytic mechanism. Subsequently, by integrating WcTre6PPase into a novel multienzyme cascade, comprising maltose phosphorylase, polyphosphate glucokinase, and AMP/ADP-polyphosphate phosphotransferase, and optimizing reaction conditions, we achieved a remarkable Tre6P yield of 90% from maltose. This study provides molecular insights into the function of WcTre6PPase and establishes a prospective platform for Tre6P production for agricultural applications.
研究领域:
结构生物学
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生物化学
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植物代谢
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酶学
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生物催化
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农业生物技术
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DOI
PubMed
高影响因子
INTERNATIONAL JOURNAL OF BIOLOGICAL MACROMOLECULES
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2024
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IF 7.7
Hao Liu,Min Huang,Shuxin Wei,Xiaowen Wang,Yaqin Zhao,Zhengyang Han,Xianfeng Ye,Zhoukun Li,Yanling Ji,Zhongli Cui,Yan Huang
β-1,3-Galactanases selectively degrade β-1,3-galactan, thus it is an attractive enzyme technique to map high-galactan structure and prepare galactooligosaccharides. In this work, a gene encoding exo -β-1,3-galactanase (PxGal43) was screened form Paenibacillus xylanexedens , consisting of a GH43 domain, a CBM32 domain and α-L-arabinofuranosidase B (AbfB) domain. Using β-1,3-galactan (AG-II-P) as substrate, the recombined enzyme expressed in Escherichia coli BL21 (DE3) exhibited an optimal activity at pH 7.0 and 30 °C. The enzyme was thermostable, retaining >70 % activity after incubating at 50 °C for 2 h. In addition, it showed high tolerance to various metal ions, denaturants and detergents. Substrate specificity indicated that PxGal43 hydrolysis only β-1,3-linked galactosyl oligosaccharides and polysaccharides, releasing galactose as an exo -acting manner. The function of the CBM32 and AbfB domain was revealed by their sequential deletion and suggested that their connection to the catalytic domain was crucial for the oligomerization, catalytic activity, substrate binding and thermal stability of PxGal43. The substrate docking and site-directed mutagenesis proposed that Glu191, Gln244, Asp138 and Glu81 served as the catalytic acid, catalytic base, pKa modulator, and substrate identifier in PxGal43, respectively. These results provide a better understanding and optimization of multi-domain bacterial GH43 β-1,3-galactanase for the degradation of arabinogalactan.
研究领域:
生物化学
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酶学
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微生物学
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DOI
PubMed
高影响因子
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