Browsing by Author "Lu, Lu-Yi"

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Dimerization Is Responsible for the Structural Stability of Human Sulfotransferase 1A1
(2009-05-01)
Lu, Lu-Yi
;
Chiang, Han-Ping
;
Chen, Wei-Ti
;
Yang, Yuh-Shyong
;
生物科技學系
Cytosolic sulfotransferases (SULTs) are responsible for the metabolism of a variety of drugs, xenobiotics, and endogenous compounds. Most cytosolic SULTs are found to be homodimers. However, transformation between monomeric and dimeric SULTs can be achieved by a single amino acid mutation. The importance of quaternary structure for cytosolic sulfotransferase was investigated using recombinant human SULT1A1, a homodimer, and its monomeric mutant (V270E). The differences between dimeric and monomeric SULT1A1 were examined by size-exclusion liquid chromatography, enzyme kinetics, substrate binding affinity, thermal inactivation, conformational stability, and circular dichroism. Variations, especially on their secondary structures and stability, between homodimer and monomer of human SULT1A1 were observed. It was found that the active site of SULT1A1 was not significantly perturbed after the change of its quaternary structure according to SULT1A1 kinetics and substrate binding affinity. However, the stability of monomeric SULT1A1 is significantly decreased. We proposed that the importance of human SULT1A1 as a homodimer was to maintain its structural stability, and the change of secondary structure was responsible for alternating its quaternary structure.
Fluorescence assay for protein post-translational tyrosine sulfation
(2013-02-01)
Chen, Bo-Han
;
Wang, Chen-Chu
;
Lu, Lu-Yi
;
Hung, Kuo-Sheng
;
Yang, Yuh-Shyong
We developed a fluorescent assay to conveniently determine the kinetics of protein sulfation, which is essential for understanding interface between protein sulfation and protein-protein interactions. Tyrosylprotein sulfotransferase (TPST) catalyzes protein sulfation using 3'-phosphate 5'-phosphosulfate (PAPS) as sulfuryl group donor. In this report, PAPS was regenerated following sulfuryl group transfer between adenosine 3',5'-diphosphate and 4-methylumbelliferyl sulfate catalyzed by phenol sulfotransferase (PST). The TPST and PST coupled enzyme platform continuously generated fluorescent 4-methylumbelliferone (MU) that was used to real-time monitor protein sulfation. Using a recombinant N utilization substance protein A fused Drosophila melanogaster tyrosylprotein sulfotransferase, we demonstrated that the activity of TPST determined through MU fluorescence directly correlated with protein sulfation. Kinetic constants obtained with small P-selectin glycoprotein ligand-1 peptide (PSGL-1 peptide, MW 1541) or its large glutathione S-transferase fusion protein (GST-PSGL-1, MW 27833) exhibited significant variation. This assay can be further developed to a high-throughput method for the characterization of TPSTs and for the identification and screening of their protein substrates.
From sequence and structure of sulfotransferases and dihydropyrimidinases to an understanding of their mechanisms of action and function
(2010-05-01)
Hsu, Ching-Chen
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Lu, Lu-Yi
;
Yang, Yuh-Shyong
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生物科技學系
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Department of Biological Science and Technology
Importance of the field: Most enzymes that catalyze physiologically important reactions are known to be highly efficient and specific to their particular substrates. On the contrary, the enzymes of detoxification are known to catalyze unlimited number of substrates containing similar function groups that may have significant structural variation. This review explores nature's strategy to design enzymes with special properties. Areas covered in this review: We review articles from 1981 to 2009, with special focus on the relationships of sequence, structure and function of cytosolic sulfotransferases and dihydropyrimidinase (DHP). What the reader will gain: Specific amino acids responsible for substrate inhibition, substrate binding orientations, substrate specificity, quaternary structures and inactivation of sulfotransferases and DHP related enzymes are elucidated. Susceptibility to some diseases possibly resulted from the mutation of a single amino acid that causes dysfunction of these enzymes. Terminal deletion of amino acid that may affect surface interaction, subunit dissociation, stability alteration and then cause the syndrome of DHP deficiency is discussed. Take home message: Based on the multiple sequence/structure analysis and with sufficient information from other members of the same enzyme families, the origin and mechanism of specific enzyme actions and proteins assembly can be clarified and predicted.
Identification and characterization of two amino acids critical for the substrate inhibition of human dehydroepiandrosterone sulfotransferase (SULT2A1)
(2008-03-01)
Lu, Lu-Yi
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Hsieh, Yin-Cheng
;
Liu, Ming-Yih
;
Lin, Yih-Hung
;
Chen, Chun-Jung
Substrate inhibition is a characteristic feature of many cytosolic sulfotransferases. The differences between the complex structures of SULT2A1/DHEA and SULT2A1/PAP or SULT2A1/ADT ( Protein Data Bank codes are 1J99, 1EFH, and 1OV4, respectively) have enabled us to elucidate the specific amino acids responsible for substrate inhibition. Based on the structural analyses, substitution of the smaller residue alanine for Tyr-238 (Y238A) significantly increases the K-i value for dehydroepiandrosterone ( DHEA) and totally eliminates substrate inhibition for androsterone (ADT). In addition, Met-137 was proposed to regulate the binding orientations of DHEA and ADT in SULT2A1. Complete elimination or regeneration of substrate inhibition for SULT2A1 with DHEA or ADT as substrate, respectively, was demonstrated with the mutations of Met-137 on Y238A mutant. Analysis of the Met-137 mutants and Met-137/Tyr-238 double mutants uncovered the relationship between substrate binding orientations and inhibition in SULT2A1. Our data indicate that, in the substrate inhibition mode, Tyr-238 regulates the release of bound substrate, and Met-137 controls substrate binding orientation of DHEA and ADT in SULT2A1. The proposed substrate inhibition mechanism is further confirmed by the crystal structures of SULT2A1 mutants at Met-137. We propose that both substrate binding orientations exhibited substrate inhibition. In addition, a corresponding residue in other cytosolic sulfotransferases was shown to have a function similar to that of Tyr-238 in SULT2A1.
Implantation of Post-translational Tyrosylprotein Sulfation into a Prokaryotic Expression System
(2011-02-11)
Lu, Lu-Yi
;
Chen, Bo-Han
;
Wu, Jennifer Yun-Shin
;
Wang, Chen-Chu
;
Chen, Da-Huang
Preparation of Tyrosylprotein Sulfotransferases for In Vitro One-Pot Enzymatic Synthesis of Sulfated Proteins/Peptides
(2018-09-01)
Wang, Chen-Chu
;
Chen, Bo-Han
;
Lu, Lu-Yi
;
Hung, Kuo-Sheng
;
Yang, Yuh-Shyong
Protein tyrosine sulfahon (PTS), catalyzed by meinbraneanchored tyrosylprotein sulfotransferase (TPST), is one of the most common post-translational modifications of secretory and transmembrane proteins. PTS, a key modulator of extracellular protein proteininteractions, accounts for various important biological activities, namely, virus entry, inflammation, coagulation, and sterility. The preparation and characterization of TPST is fundamental for understanding the synthesis of tyrosine-sulfated proteins and for studying PTS in biology. A sulfated protein was prepared using a TPSTT coupled protein sulfation system that involves the generation of the active sulfate 31-phosphoadenosine-51-phosphosulfate (PAPS) through either PAPS synthetase (PAPSS) or phenol sulfotransferase. The preparation of sulfated proteins was confirmed through radiometric or immunochemical assays. In this study, enzymatically active Drosophila melanogaster TPST (DruTPST) and human TPSTs (hTPST1 and hTPST2) were expressed in Escherichia colt BL21(DE3) host cells and purified to homogeneity in high yield. Our results revealed that recombinant DmTPST was particularly useful considering its catalytic efficiency and ease of preparation in large quantities. This study provides tools for high-efficiency, one-step synthesis of sulfated proteim arid peptides that are useful for further deciphering the mechanisms, functions, and future applications of PTS.
Spectrofluorometric assay for monoamine-preferring phenol sulfotransferase (SULT1A3)
(2010-09-15)
Lu, Lu-Yi
;
Hsu, Yen-Chang
;
Yang, Yuh-Shyong
;
生物科技學系
;
Department of Biological Science and Technology
A continuous and real-time fluorometric assay for monoamine-preferring phenol sulfotransferase (SULT1A3) was developed. The methodology was based on the coupling of SULT1A1 to regenerate 3'-phosphoadenosine-5'-phosPhosulfate (PAPS) using 4-methylumbelliferyl sulfate (MUS) as a sulfuryl group donor. The fluorophore product (4-methylumbelliferone. MU) was continuously produced and monitored when SULT1A3 catalyzed dopamine sulfation with PAPS. The optimal conditions of this turnover reaction and substrate inhibition of SULT1A3 were also determined. This coupled-enzyme assay allows the continuous measurement of initial reaction velocity and the sensitivity is comparable to that of end-point radioactive isotope assay. (C) 2010 Elsevier Inc. All rights reserved.