Honors & Awards
Postdoctoral fellowship from Taiwan Bio-Development Foundation., Taiwan Bio-Development Foundation (2015-2017)
Academia Sinica distinguished postdoctoral scholar., Academia Sinica (2014-2015)
Outstanding performance for the 2014 19th biophysics conference., Biophysical Society of Taiwan (2014)
Selected young scientist oral presentation for the 2014 NSRRC 20th Users’ Meeting & Workshops., National Synchrotron Radiation Research Center (NSRRC) (2014)
Outstanding student research award, 2nd GRC research staff award., Genomics Research Center (GRC), Academia Sinica (2012)
TIGP student conference travel grant award., Taiwan International Graduate Program (TIGP), Academia Sinica (2012)
Outstanding performance for the 2011 16th biophysics conference., Biophysical Society of Taiwan (2011)
Outstanding performance for the 2011 NSRRC 17th Users’ Meeting & Workshop., National Synchrotron Radiation Research Center (NSRRC) (2011)
Outstanding performance for the 2011 Symposium on Frontiers of Biomedical Sciences., Prof. Jung-Yaw Lin Science and Education Foundation (2011)
Outstanding student research award, 1st GRC research staff award., Genomics Research Center (GRC), Academia Sinica (2011)
Outstanding student research award, 21st Wang Ming-Ning Memorial Foundation award., Wang Ming-Ning Memorial Foundation (2011)
Outstanding student research award, College of Life Science, National Taiwan University., National Taiwan University (2011)
Outstanding performance for the 2010 CBMB, IBC, and IBS retreat., Taiwan International Graduate Program (TIGP), Academia Sinica (2010)
Doctor of Philosophy, National Taiwan University (2013)
Master of Science, National Cheng Kung University (2004)
Bachelor of Chemistry, National Cheng Kung University (2002)
Tsung-Lin Li, Syue-Yi Lyu, Yu-Chen Liu, Chin-Yuan Chang. "Taiwan Patent I 561532 Teicoplanin analogs and uses thereof", Academia Sinica, Dec 11, 2016
Tsung-Lin Li, Yu-Chen Liu, Yi-Shan Li, Syue-Yi Lyu. "Taiwan Patent I 462743 Structural and mechanistic basis for novel compound biosynthesis using the 4-electron hexose oxidase.", Academia Sinica, Dec 1, 2014
Tsung-Lin Li, Yu-Chen Liu, Yi-Shan Li, Syue-Yi Lyu. "United States Patent US 8,951,961 B2 Structural and mechanistic basis for novel compound biosynthesis using the 4-electron hexose oxidase-extension.", Academia Sinica, Oct 31, 2013
Tsung-Lin Li, Yu-Chen Liu, Yi-Shan Li, Syue-Yi Lyu. "United States Patent US 8,507,427 B2 Structural and mechanistic basis for novel compound biosynthesis using the 4-electron hexose oxidase.", Academia Sinica, May 3, 2012
Structure and mechanism of assembly line polyketide synthases.
Current opinion in structural biology
2016; 41: 10-18
Assembly line polyketide synthases (PKSs) are remarkable biosynthetic machines with considerable potential for structure-based engineering. Several types of protein-protein interactions, both within and between PKS modules, play important roles in the catalytic cycle of a multimodular PKS. Additionally, vectorial biosynthesis is enabled by the energetic coupling of polyketide chain elongation to the channeling of intermediates between successive modules. A combination of high-resolution analysis of smaller PKS components and lower resolution characterization of intact modules and bimodules has yielded insights into the structure and organization of a prototypical assembly line PKS. This review discusses our understanding of key structure-function relationships in this family of megasynthases, along with a recap of key unanswered questions in the field.
View details for DOI 10.1016/j.sbi.2016.05.009
View details for PubMedID 27266330
Multiple Complexes of Long Aliphatic N-Acyltransferases Lead to Synthesis of 2,6-Diacylated/2-Acyl-Substituted Glycopeptide Antibiotics, Effectively Killing Vancomycin-Resistant Enterococcus
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY
2014; 136 (31): 10989-10995
Teicoplanin A2-2 (Tei)/A40926 is the last-line antibiotic to treat multidrug-resistant Gram-positive bacterial infections, e.g., methicillin-resistant Staphylococcus aureus (MRSA) and vancomycin-resistant enterococcus (VRE). This class of antibiotics is powered by the N-acyltransferase (NAT) Orf11*/Dbv8 through N-acylation on glucosamine at the central residue of Tei/A40926 pseudoaglycone. The NAT enzyme possesses enormous value in untapped applications; its advanced development is hampered largely due to a lack of structural information. In this report, we present eight high-resolution X-ray crystallographic unary, binary, and ternary complexes in order to decipher the molecular basis for NAT's functionality. The enzyme undergoes a multistage conformational change upon binding of acyl-CoA, thus allowing the uploading of Tei pseudoaglycone to enable the acyl-transfer reaction to take place in the occlusion between the N- and C-halves of the protein. The acyl moiety of acyl-CoA can be bulky or lengthy, allowing a large extent of diversity in new derivatives that can be formed upon its transfer. Vancomycin/synthetic acyl-N-acetyl cysteamine was not expected to be able to serve as a surrogate for an acyl acceptor/donor, respectively. Most strikingly, NAT can catalyze formation of 2-N,6-O-diacylated or C6→C2 acyl-substituted Tei analogues through an unusual 1,4-migration mechanism under stoichiometric/solvational reaction control, wherein selected representatives showed excellent biological activities, effectively counteracting major types (VanABC) of VRE.
View details for DOI 10.1021/ja504125v
View details for Web of Science ID 000340079800034
View details for PubMedID 25095906
Structure and mechanism of a nonhaem-iron SAM-dependent C-methyltransferase and its engineering to a hydratase and an O-methyltransferase
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
2014; 70: 1549-1560
In biological systems, methylation is most commonly performed by methyltransferases (MTs) using the electrophilic methyl source S-adenosyl-L-methionine (SAM) via the S(N)2 mechanism. (2S,3S)-β-Methylphenylalanine, a nonproteinogenic amino acid, is a building unit of the glycopeptide antibiotic mannopeptimycin. The gene product of mppJ from the mannopeptimycin-biosynthetic gene cluster is the MT that methylates the benzylic C atom of phenylpyruvate (Ppy) to give βMePpy. Although the benzylic C atom of Ppy is acidic, how its nucleophilicity is further enhanced to become an acceptor for C-methylation has not conclusively been determined. Here, a structural approach is used to address the mechanism of MppJ and to engineer it for new functions. The purified MppJ displays a turquoise colour, implying the presence of a metal ion. The crystal structures reveal MppJ to be the first ferric ion SAM-dependent MT. An additional four structures of binary and ternary complexes illustrate the molecular mechanism for the metal ion-dependent methyltransfer reaction. Overall, MppJ has a nonhaem iron centre that bind, orients and activates the α-ketoacid substrate and has developed a sandwiched bi-water device to avoid the formation of the unwanted reactive oxo-iron(IV) species during the C-methylation reaction. This discovery further prompted the conversion of the MT into a structurally/functionally unrelated new enzyme. Through stepwise mutagenesis and manipulation of coordination chemistry, MppJ was engineered to perform both Lewis acid-assisted hydration and/or O-methyltransfer reactions to give stereospecific new compounds. This process was validated by six crystal structures. The results reported in this study will facilitate the development and design of new biocatalysts for difficult-to-synthesize biochemicals.
View details for DOI 10.1107/S1399004714005239
View details for Web of Science ID 000338117800006
View details for PubMedID 24914966
Insights into the binding specificity and catalytic mechanism of N-acetylhexosamine 1-phosphate kinases through multiple reaction complexes
ACTA CRYSTALLOGRAPHICA SECTION D-BIOLOGICAL CRYSTALLOGRAPHY
2014; 70: 1401-1410
Utilization of N-acetylhexosamine in bifidobacteria requires the specific lacto-N-biose/galacto-N-biose pathway, a pathway differing from the Leloir pathway while establishing symbiosis between humans and bifidobacteria. The gene lnpB in the pathway encodes a novel hexosamine kinase NahK, which catalyzes the formation of N-acetylhexosamine 1-phosphate (GlcNAc-1P/GalNAc-1P). In this report, seven three-dimensional structures of NahK in complex with GlcNAc, GalNAc, GlcNAc-1P, GlcNAc/AMPPNP and GlcNAc-1P/ADP from both Bifidobacterium longum (JCM1217) and B. infantis (ATCC15697) were solved at resolutions of 1.5-2.2 Å. NahK is a monomer in solution, and its polypeptide folds in a crescent-like architecture subdivided into two domains by a deep cleft. The NahK structures presented here represent the first multiple reaction complexes of the enzyme. This structural information reveals the molecular basis for the recognition of the given substrates and products, GlcNAc/GalNAc, GlcNAc-1P/GalNAc-1P, ATP/ADP and Mg(2+), and provides insights into the catalytic mechanism, enabling NahK and mutants thereof to form a choice of biocatalysts for enzymatic and chemoenzymatic synthesis of carbohydrates.
View details for DOI 10.1107/S1399004714004209
View details for Web of Science ID 000335952500021
View details for PubMedID 24816108
Biosynthesis of Streptolidine Involved Two Unexpected Intermediates Produced by a Dihydroxylase and a Cyclase through Unusual Mechanisms
ANGEWANDTE CHEMIE-INTERNATIONAL EDITION
2014; 53 (7): 1943-1948
Streptothricin-F (STT-F), one of the early-discovered antibiotics, consists of three components, a β-lysine homopolymer, an aminosugar D-gulosamine, and an unusual bicyclic streptolidine. The biosynthesis of streptolidine is a long-lasting but unresolved puzzle. Herein, a combination of genetic/biochemical/structural approaches was used to unravel this problem. The STT gene cluster was first sequenced from a Streptomyces variant BCRC 12163, wherein two gene products OrfP and OrfR were characterized in vitro to be a dihydroxylase and a cyclase, respectively. Thirteen high-resolution crystal structures for both enzymes in different reaction intermediate states were snapshotted to help elucidate their catalytic mechanisms. OrfP catalyzes an Fe(II) -dependent double hydroxylation reaction converting L-Arg into (3R,4R)-(OH)2 -L-Arg via (3S)-OH-L-Arg, while OrfR catalyzes an unusual PLP-dependent elimination/addition reaction cyclizing (3R,4R)-(OH)2 -L-Arg to the six-membered (4R)-OH-capreomycidine. The biosynthetic mystery finally comes to light as the latter product was incorporation into STT-F by a feeding experiment.
View details for DOI 10.1002/anie.201307989
View details for Web of Science ID 000330680400036
View details for PubMedID 24505011
Chain Elongation and Cyclization in Type III PKS DpgA
2012; 13 (6): 862-871
Chain elongation and cyclization of precursors of dihydroxyphenylacetyl-CoA (DPA-CoA) catalyzed by the bacterial type III polyketide synthase DpgA were studied. Two labile intermediates, di- and tri-ketidyl-CoA (DK- and TK-CoA), were proposed and chemically synthesized. In the presence of DpgABD, each of these with [(13)C(3)]malonyl-CoA (MA-CoA) was able to form partially (13)C-enriched DPA-CoA. By NMR and MS analysis, the distribution of (13)C atoms in the partially (13)C-enriched DPA-CoA shed light on how the polyketide chain elongates and cyclizes in the DpgA-catalyzed reaction. Polyketone intermediates elongate in a manner different from that which had been believed: two molecules of DK-CoA, or one DK-CoA plus one acetoacetyl-CoA (AA-CoA), but not two molecules of AA-CoA can form one molecule of DPA-CoA. As a result, polyketidyl-CoA serves as both the starter and extender, whereas polyketone-CoA without the terminal carboxyl group can only act as an extender. The terminal carboxyl group is crucial for the cyclization that likely takes place on CoA.
View details for DOI 10.1002/cbic.201200051
View details for Web of Science ID 000302609100016
View details for PubMedID 22492619
Combining biocatalysis and chemoselective chemistries for glycopeptide antibiotics modification
CURRENT OPINION IN CHEMICAL BIOLOGY
2012; 16 (1-2): 170-178
Glycopeptide antibiotics are clinically important medicines to treat serious Gram-positive bacterial infections. The emergence of glycopeptide resistance among pathogens has motivated considerable interest in expanding structural diversity of glycopeptide to counteract resistance. The complex structure of glycopeptide poses substantial barriers to conventional chemical methods for structural modifications. By contrast, biochemical approaches have attracted great attention because ample biosynthetic information and sophisticated toolboxes have been made available to change reaction specificity through protein engineering, domain swapping, pathway engineering, addition of substrate analogs, and mutagenesis.
View details for DOI 10.1016/j.cbpa.2012.01.017
View details for Web of Science ID 000303640400023
View details for PubMedID 22336892
Interception of teicoplanin oxidation intermediates yields new antimicrobial scaffolds
NATURE CHEMICAL BIOLOGY
2011; 7 (5): 304-309
In the search for new efficacious antibiotics, biosynthetic engineering offers attractive opportunities to introduce minor alterations to antibiotic structures that may overcome resistance. Dbv29, a flavin-containing oxidase, catalyzes the four-electron oxidation of a vancomycin-like glycopeptide to yield A40926. Structural and biochemical examination of Dbv29 now provides insights into residues that govern flavinylation and activity, protein conformation and reaction mechanism. In particular, the serendipitous discovery of a reaction intermediate in the crystal structure led us to identify an unexpected opportunity to intercept the normal enzyme mechanism at two different points to create new teicoplanin analogs. Using this method, we synthesized families of antibiotic analogs with amidated and aminated lipid chains, some of which showed marked potency and efficacy against multidrug resistant pathogens. This method offers a new strategy for the development of chemical diversity to combat antibacterial resistance.
View details for DOI 10.1038/nchembio.556
View details for Web of Science ID 000289617800014
View details for PubMedID 21478878
Regioselective deacetylation based on teicoplanin-complexed Orf2*crystal structures
2011; 7 (4): 1224-1231
Lipoglycopeptide antibiotics are more effective than vancomycin against MRSA as they carry an extra aliphatic acyl side chain on glucosamine (Glm) at residue 4 (r4). The biosynthesis of the r4 N-acyl Glc moiety at teicoplanin (Tei) or A40926 has been elucidated, in which the primary amine nucleophile of Glm is freed from the r4 GlcNac pseudo-Tei precursor by Orf2* for the subsequent acylation reaction to occur. In this report, two Orf2* structures in complex with β-D-octyl glucoside or Tei were solved. Of the complexed structures, the substrate binding site and a previously unknown hydrophobic cavity were revealed, wherein r4 GlcNac acts as the key signature for molecular recognition and the cavity allows substrates carrying longer acyl side chains in addition to the acetyl group. On the basis of the complexed structures, a triple-mutation mutant S98A/V121A/F193Y is able to regioselectively deacetylate r6 GlcNac pseudo-Tei instead of that at r4. Thereby, novel analogs can be made at the r6 sugar moiety.
View details for DOI 10.1039/c0mb00320d
View details for Web of Science ID 000288329300030
View details for PubMedID 21267472
In vitro Characterization of Enzymes Involved in the Synthesis of Nonproteinogenic Residue (2S,3S)-beta-Methylphenylalanine in Glycopeptide Antibiotic Mannopeptimycin
2009; 10 (15): 2480-2487
Mannopeptimycin, a potent drug lead, has superior activity against difficult-to-treat multidrug-resistant Gram-positive pathogens such as methicillin-resistant Staphylococcus aureus (MRSA). (2S,3S)-beta-Methylphenylalanine is a residue in the cyclic hexapeptide core of mannopeptimycin, but the synthesis of this residue is far from clear. We report here on the reaction order and the stereochemical course of reaction in the formation of (2S,3S)-beta-methylphenylalanine. The reaction is executed by the enzymes MppJ and TyrB, an S-adenosyl methionine (SAM)-dependent methyltransferase and an (S)-aromatic-amino-acid aminotransferase, respectively. Phenylpyruvic acid is methylated by MppJ at its benzylic position at the expense of one equivalent of SAM. The resulting beta-methyl phenylpyruvic acid is then converted to (2S,3S)-beta-methylphenylalanine by TyrB. MppJ was further determined to be regioselective and stereoselective in its catalysis of the formation of (3S)-beta-methylphenylpyruvic acid. The binding constant (K(D)) of MppJ versus SAM is 26 microM. The kinetic constants with respect to k(cat Ppy) and K(M Ppy), and k(cat SAM) and K(M SAM) are 0.8 s(-1) and 2.5 mM, and 8.15 s(-1) and 0.014 mM, respectively. These results suggest SAM has higher binding affinity for MppJ than Ppy, and the C--C bond formation in betamPpy might be the rate-limiting step, as opposed to the C--S bond breakage in SAM.
View details for DOI 10.1002/cbic.200900351
View details for Web of Science ID 000271095600009
View details for PubMedID 19731276
Effect of D To E mutation of the RGD motif in rhodostomin on its activity, structure, and dynamics: Importance of the interactions between the D residue and integrin
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
2009; 76 (4): 808-821
Rhodostomin (Rho) is a snake venom protein containing an RGD motif that specifically inhibits the integrin-binding function. Rho produced in Pichia pastoris inhibits platelet aggregation with a K(I) of 78 nM as potent as native Rho. In contrast, its D51E mutant inhibits platelet aggregation with a K(I) of 49 muM. Structural analysis of Rho and its D51E mutant showed that they have the same tertiary fold with three two-stranded antiparallel beta-sheets. There are no structural backbone differences between the RG[D/E] loop which extends outward from the protein core and the RG[D/E] sequence at its apex in a four-residue RG[D/E]M type I turn. Two minor differences between Rho and its D51E mutant were only found from their backbone dynamics and 3D structures. The R(2) value of E51 is 13% higher than that of the D51 residue. A difference in the charge separation of 1.76 A was found between the sidechains of positive (R49) and negative residues (D51 or E51).The docking of Rho into integrin alphavbeta3 showed that the backbone amide and carbonyl groups of the D51 residue of Rho were formed hydrogen bonds with the integrin residues R216 and R214, respectively. In contrast, these hydrogen bonds were absent in the D51E mutant-integrin complex. Our findings suggest that the interactions between both the sidechain and backbone of the D residue of RGD-containing ligands and integrin are important for their binding.
View details for DOI 10.1002/prot.22387
View details for Web of Science ID 000268839400004
View details for PubMedID 19280603
Solution structure of gamma-bungarotoxin: The functional significance of amino acid residues flanking the RGD motif in integrin binding
PROTEINS-STRUCTURE FUNCTION AND BIOINFORMATICS
2004; 57 (4): 839-849
Gamma-bungarotoxin, a snake venom protein isolated from Bungarus multicinctus, contains 68 amino acids, including 10 cysteine residues and a TAVRGDGP sequence at positions 30-37. The solution structure of gamma-bungarotoxin has been determined by nuclear magnetic resonance (NMR) spectroscopy. The structure is similar to that of the short-chain neurotoxins that contain three loops extending from a disulfide-bridged core. The tripeptide Arg-Gly-Asp (RGD) sequence is located at the apex of the flexible loop and is similar to that of other RGD-containing proteins. However, gamma-bungarotoxin only inhibits platelet aggregations with an IC50 of 34 microM. To understand its weak activity in inhibiting platelet aggregation, we mutated the RGD loop sequences of rhodostomin, a potent platelet aggregation inhibitor, from RIPRGDMP to TAVRGDGP, resulting in a 196-fold decrease in activity. In addition, the average Calpha-to-Calpha distance between R33 and G36 of gamma-bungarotoxin is 6.02 A, i.e., shorter than that of other RGD-containing proteins that range from 6.55 to 7.46 A. These results suggested that the amino acid residues flanking the RGD motif might control the width of the RGD loop. This structural difference may be responsible for its decrease in platelet aggregation inhibition compared with other RGD-containing proteins.
View details for DOI 10.1002/prot.20269
View details for Web of Science ID 000225351100021
View details for PubMedID 15390258