Publications by authors named "Tzu-Ping Ko"

173 Publications

Crystal structure and functional implication of a bacterial cyclic AMP-AMP-GMP synthetase.

Nucleic Acids Res 2021 May;49(8):4725-4737

Institute of New Drug Development, China Medical University, Taichung 406, Taiwan.

Mammalian cyclic GMP-AMP synthase (cGAS) and its homologue dinucleotide cyclase in Vibrio cholerae (VcDncV) produce cyclic dinucleotides (CDNs) that participate in the defense against viral infection. Recently, scores of new cGAS/DncV-like nucleotidyltransferases (CD-NTases) were discovered, which produce various CDNs and cyclic trinucleotides (CTNs) as second messengers. Here, we present the crystal structures of EcCdnD, a CD-NTase from Enterobacter cloacae that produces cyclic AMP-AMP-GMP, in its apo-form and in complex with ATP, ADP and AMPcPP, an ATP analogue. Despite the similar overall architecture, the protein shows significant structural variations from other CD-NTases. Adjacent to the donor substrate, another nucleotide is bound to the acceptor binding site by a non-productive mode. Isothermal titration calorimetry results also suggest the presence of two ATP binding sites. GTP alone does not bind to EcCdnD, which however binds to pppApG, a possible intermediate. The enzyme is active on ATP or a mixture of ATP and GTP, and the best metal cofactor is Mg2+. The conserved residues Asp69 and Asp71 are essential for catalysis, as indicated by the loss of activity in the mutants. Based on structural analysis and comparison with VcDncV and RNA polymerase, a tentative catalytic pathway for the CTN-producing EcCdnD is proposed.
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http://dx.doi.org/10.1093/nar/gkab165DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8096243PMC
May 2021

A Unique Carboxylic-Acid Hydrogen-Bond Network (CAHBN) Confers Glutaminyl Cyclase Activity on M28 Family Enzymes.

J Mol Biol 2021 Mar 25;433(13):166960. Epub 2021 Mar 25.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan. Electronic address:

Proteins with sequence or structure similar to those of di-Zn exopeptidases are usually classified as the M28-family enzymes, including the mammalian-type glutaminyl cyclases (QCs). QC catalyzes protein N-terminal pyroglutamate formation, a posttranslational modification important under many physiological and pathological conditions, and is a drug target for treating neurodegenerative diseases, cancers and inflammatory disorders. Without functional characterization, mammalian QCs and their orthologs remain indistinguishable at the sequence and structure levels from other M28-family proteins, leading to few reported QCs. Here, we show that a low-barrier carboxylic-acid hydrogen-bond network (CAHBN) is required for QC activity and discriminates QCs from M28-family peptidases. We demonstrate that the CAHBN-containing M28 peptidases deposited in the PDB are indeed QCs. Our analyses identify several thousands of QCs from the three domains of life, and we enzymatically and structurally characterize several. For the first time, the interplay between a CAHBN and the binuclear metal-binding center of mammalian QCs is made clear. We found that the presence or absence of CAHBN is a key discriminator for the formation of either the mono-Zn QCs or the di-Zn exopeptidases. Our study helps explain the possible roles of QCs in life.
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http://dx.doi.org/10.1016/j.jmb.2021.166960DOI Listing
March 2021

Crystal structure of the N-terminal domain of TagH reveals a potential drug targeting site.

Biochem Biophys Res Commun 2021 01 22;536:1-6. Epub 2020 Dec 22.

Institute of New Drug Development, China Medical University, Taichung, 406, Taiwan; Drug Development Center, Research Center for Cancer Biology and Center for Molecular Medicine, China Medical University, Taichung, 40402, Taiwan. Electronic address:

Bacterial wall teichoic acids (WTAs) are synthesized intracellularly and exported by a two-component transporter, TagGH, comprising the transmembrane and ATPase subunits TagG and TagH. Here the dimeric structure of the N-terminal domain of TagH (TagH-N) was solved by single-wavelength anomalous diffraction using a selenomethionine-containing crystal, which shows an ATP-binding cassette (ABC) architecture with RecA-like and helical subdomains. Besides significant structural differences from other ABC transporters, a prominent patch of positively charged surface is seen in the center of the TagH-N dimer, suggesting a potential binding site for the glycerol phosphate chain of WTA. The ATPase activity of TagH-N was inhibited by clodronate, a bisphosphonate, in a non-competitive manner, consistent with the proposed WTA-binding site for drug targeting.
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http://dx.doi.org/10.1016/j.bbrc.2020.12.028DOI Listing
January 2021

Cloning, expression, identification and characterization of borneol dehydrogenase isozymes in Pseudomonas sp. TCU-HL1.

Protein Expr Purif 2020 11 29;175:105715. Epub 2020 Jul 29.

Department of Biochemistry, Tzu Chi University, Hualien, 97004, Taiwan; Institute of Medical Sciences, School of Medicine, Tzu Chi University, Hualien, 97004, Taiwan; Integration Center of Traditional Chinese and Modern Medicine, Hualien Tzu Chi Hospital, Hualien, 97002, Taiwan. Electronic address:

Borneol is a bicyclic plant monoterpene. It can be degraded by soil microorganisms through the conversion of borneol dehydrogenase (BDH) and a known camphor degradation pathway. Recombinant BDH from Pseudomonas sp. TCU-HL1 was produced in the form of inclusion body. The refolded BDH1 tends to precipitate. Insoluble recombinant BDH1 was converted into a soluble form by adding glycerol in LB medium. The k and k/K values of soluble form BDH1 for (+)-borneol turned out to be about 34-fold and 45-fold higher, respectively, than those of the refolded enzyme. On the other hand, a gene knockout mutant, TCU-HL1Δbdh, was constructed to investigate the possible presence of a second copy of the bdh gene in TCU-HL1 genome. A new gene, bdh2, encoding a BDH isozyme, was identified, and the recombinant BDH2 protein was produced in a soluble form. Both bdh1 and bdh2 genes are expressed in the crude extract of wild type TCU-HL1, as shown by RT-qPCR results. Both BDH isozymes prefer to degrade (+)-borneol, rather than (-)-borneol, probably because (+)-camphor is the main form present in nature.
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http://dx.doi.org/10.1016/j.pep.2020.105715DOI Listing
November 2020

Structural insights into thebaine synthase 2 catalysis.

Biochem Biophys Res Commun 2020 08 22;529(2):156-161. Epub 2020 Jun 22.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, PR China. Electronic address:

Thebaine synthase 2 (THS2) that can transform (7S)-salutaridinol 7-O-acetate to thebaine catalyzes the final step of thebaine biosynthesis in Papaver somniferum. Here, the crystal structures of THS2 and its complex with thebaine are reported, revealing the interaction network in the substrate-binding pocket. Subsequent docking and QM/MM studies was performed to further explore the catalytic mechanism of THS2. Our results suggest that T105 may abstract the proton of C4-OH from the substrate under the assistance of H89. The resulting C4-O phenolate anion then attacks the nearby C5, and triggers intramolecular S2' syn displacement with the elimination of O-acetyl group. Moreover, the latter S2' reaction is the rate-determining step of the whole enzymatic reaction with an overall energy barrier of 18.8 kcal/mol. These findings are of pivotal importance to understand the mechanism of action of thebaine biosynthesis, and would guide enzyme engineering to enhance the production of opiate alkaloids via metabolic engineering.
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http://dx.doi.org/10.1016/j.bbrc.2020.05.199DOI Listing
August 2020

Structural characterization of borneol dehydrogenase from Pseudomonas sp. TCU-HL1.

Acta Crystallogr F Struct Biol Commun 2020 Jul 1;76(Pt 7):309-313. Epub 2020 Jul 1.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan.

During the microbial degradation of borneol, a bicyclic plant monoterpene, it is first converted into camphor by borneol dehydrogenase (BDH) and then enters a known camphor-degradation pathway. Previously, a recombinant Pseudomonas BDH was found in inclusion bodies when expressed in Escherichia coli. After refolding, it was still unstable and was difficult to concentrate. Here, the protein-expression conditions were improved by changing the medium from lysogeny broth to Terrific Broth, yielding a soluble form of the enzyme with higher activity. The protein was crystallized and its 3D structure was determined by X-ray diffraction. Like other known homologues such as quinuclidinone reductase, the protein forms a tetramer with subunits containing Rossmann folds. Structural comparison revealed major differences in the C-terminal helices and the associated loops. It is likely that these regions contain the determinants for substrate recognition.
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http://dx.doi.org/10.1107/S2053230X20008584DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7336358PMC
July 2020

Structural insight into the differential interactions between the DNA mimic protein SAUGI and two gamma herpesvirus uracil-DNA glycosylases.

Int J Biol Macromol 2020 Oct 3;160:903-914. Epub 2020 Jun 3.

The PhD Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 115, Taiwan; Graduate Institute of Translational Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei 110, Taiwan. Electronic address:

Uracil-DNA glycosylases (UDGs) are conserved DNA-repair enzymes that can be found in many species, including herpesviruses. Since they play crucial roles for efficient viral DNA replication in herpesviruses, they have been considered as potential antiviral targets. In our previous work, Staphylococcus aureus SAUGI was identified as a DNA mimic protein that targets UDGs from S. aureus, human, Herpes simplex virus (HSV) and Epstein-Barr virus (EBV). Interestingly, SAUGI has the strongest inhibitory effects with EBVUDG. Here, we determined complex structures of SAUGI with EBVUDG and another γ-herpesvirus UDG from Kaposi's sarcoma-associated herpesvirus (KSHVUDG), which SAUGI fails to effectively inhibit. Structural analysis of the SAUGI/EBVUDG complex suggests that the additional interaction between SAUGI and the leucine loop may explain why SAUGI shows the highest binding capacity with EBVUDG. In contrast, SAUGI appears to make only partial contacts with the key components responsible for the compression and stabilization of the DNA backbone in the leucine loop extension of KSHVUDG. The findings in this study provide a molecular explanation for the differential inhibitory effects and binding strengths that SAUGI has on these two UDGs, and the structural basis of the differences should be helpful in developing inhibitors that would interfere with viral DNA replication.
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http://dx.doi.org/10.1016/j.ijbiomac.2020.05.267DOI Listing
October 2020

Structural insight into the electron transfer pathway of a self-sufficient P450 monooxygenase.

Nat Commun 2020 05 29;11(1):2676. Epub 2020 May 29.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, P. R. China.

Cytochrome P450 monooxygenases are versatile heme-thiolate enzymes that catalyze a wide range of reactions. Self-sufficient cytochrome P450 enzymes contain the redox partners in a single polypeptide chain. Here, we present the crystal structure of full-length CYP116B46, a self-sufficient P450. The continuous polypeptide chain comprises three functional domains, which align well with the direction of electrons traveling from FMN to the heme through the [2Fe-2S] cluster. FMN and the [2Fe-2S] cluster are positioned closely, which facilitates efficient electron shuttling. The edge-to-edge straight-line distance between the [2Fe-2S] cluster and heme is approx. 25.3 Å. The role of several residues located between the [2Fe-2S] cluster and heme in the catalytic reaction is probed in mutagenesis experiments. These findings not only provide insights into the intramolecular electron transfer of self-sufficient P450s, but are also of interest for biotechnological applications of self-sufficient P450s.
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http://dx.doi.org/10.1038/s41467-020-16500-5DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7260179PMC
May 2020

Structure of a gut microbial diltiazem-metabolizing enzyme suggests possible substrate binding mode.

Biochem Biophys Res Commun 2020 06 16;527(3):799-804. Epub 2020 May 16.

School of Life Science, University of Science and Technology of China, Anhui, 230026, China; Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, China. Electronic address:

When administrated orally, the vasodilating drug diltiazem can be metabolized into diacetyl diltiazem in the presence of Bacteroides thetaiotaomicron, a human gut microbe. The removal of acetyl group from the parent drug is carried out by the GDSL/SGNH-family hydrolase BT4096. Here the crystal structure of the enzyme was solved by mercury soaking and single-wavelength anomalous diffraction. The protein folds into two parts. The N-terminal part comprises the catalytic domain which is similar to other GDSL/SGNH hydrolases. The flanking C-terminal part is made up of a β-barrel subdomain and an α-helical subdomain. Structural comparison shows that the catalytic domain is most akin to acetyl-xylooligosaccharide esterase and allows a plausible binding mode of diltiazem to be proposed. The β-barrel subdomain is similar in topology to the immunoglobulin-like domains, including some carbohydrate-binding modules, of various bacterial glycoside hydrolases. Consequently, BT4096 might originally function as an oligosaccharide deacetylase with additional subdomains that could enhance substrate binding, and it acts on diltiazem just by accident.
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http://dx.doi.org/10.1016/j.bbrc.2020.04.116DOI Listing
June 2020

Structural basis of polyethylene glycol recognition by antibody.

J Biomed Sci 2020 Jan 7;27(1):12. Epub 2020 Jan 7.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Background: Polyethylene glycol (PEG) is widely used in industry and medicine. Anti-PEG antibodies have been developed for characterizing PEGylated drugs and other applications. However, the underlying mechanism for specific PEG binding has not been elucidated.

Methods: The Fab of two cognate anti-PEG antibodies 3.3 and 2B5 were each crystallized in complex with PEG, and their structures were determined by X-ray diffraction. The PEG-Fab interactions in these two crystals were analyzed and compared with those in a PEG-containing crystal of an unrelated anti-hemagglutinin 32D6-Fab. The PEG-binding stoichiometry was examined by using analytical ultracentrifuge (AUC).

Results: A common PEG-binding mode to 3.3 and 2B5 is seen with an S-shaped core PEG fragment bound to two dyad-related Fab molecules. A nearby satellite binding site may accommodate parts of a longer PEG molecule. The core PEG fragment mainly interacts with the heavy-chain residues D31, W33, L102, Y103 and Y104, making extensive contacts with the aromatic side chains. At the center of each half-circle of the S-shaped PEG, a water molecule makes alternating hydrogen bonds to the ether oxygen atoms, in a similar configuration to that of a crown ether-bound lysine. Each satellite fragment is clamped between two arginine residues, R52 from the heavy chain and R29 from the light chain, and also interacts with several aromatic side chains. In contrast, the non-specifically bound PEG fragments in the 32D6-Fab crystal are located in the elbow region or at lattice contacts. The AUC data suggest that 3.3-Fab exists as a monomer in PEG-free solution but forms a dimer in the presence of PEG-550-MME, which is about the size of the S-shaped core PEG fragment.

Conclusions: The differing amino acids in 3.3 and 2B5 are not involved in PEG binding but engaged in dimer formation. In particular, the light-chain residue K53 of 2B5-Fab makes significant contacts with the other Fab in a dimer, whereas the corresponding N53 of 3.3-Fab does not. This difference in the protein-protein interaction between two Fab molecules in a dimer may explain the temperature dependence of 2B5 in PEG binding, as well as its inhibition by crown ether.
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http://dx.doi.org/10.1186/s12929-019-0589-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6945545PMC
January 2020

Cryo-EM analysis of a feline coronavirus spike protein reveals a unique structure and camouflaging glycans.

Proc Natl Acad Sci U S A 2020 01 3;117(3):1438-1446. Epub 2020 Jan 3.

Institute of Biological Chemistry, Academia Sinica, Taipei 11529, Taiwan;

Feline infectious peritonitis virus (FIPV) is an alphacoronavirus that causes a nearly 100% mortality rate without effective treatment. Here we report a 3.3-Å cryoelectron microscopy (cryo-EM) structure of the serotype I FIPV spike (S) protein, which is responsible for host recognition and viral entry. Mass spectrometry provided site-specific compositions of densely distributed high-mannose and complex-type Nglycans that account for 1/4 of the total molecular mass; most of the N-glycans could be visualized by cryo-EM. Specifically, the N-glycans that wedge between 2 galectin-like domains within the S1 subunit of FIPV S protein result in a unique propeller-like conformation, underscoring the importance of glycosylation in maintaining protein structures. The cleavage site within the S2 subunit responsible for activation also showed distinct structural features and glycosylation. These structural insights provide a blueprint for a better molecular understanding of the pathogenesis of FIP.
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http://dx.doi.org/10.1073/pnas.1908898117DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983407PMC
January 2020

Dietary Flavonoids Luteolin and Quercetin Inhibit Migration and Invasion of Squamous Carcinoma through Reduction of Src/Stat3/S100A7 Signaling.

Antioxidants (Basel) 2019 Nov 15;8(11). Epub 2019 Nov 15.

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11034, Taiwan.

Flavonoids are well-known antioxidants and have shown the ability to prevent tumor formation and recurrence. Especially in dietary flavonoids, they have provided convenience and consistence of intake for long-term prevention of tumor formation. Previous reports suggested that S100 calcium-binding protein A7 (S100A7) might activate epithelial-mesenchymal transition (EMT) signaling and promote the metastasis of tumor cells; however, the regulatory signaling was unclear. In this study, we found that S100A7 was highly expressed in cancer cells and could be reduced by luteolin (Lu) and quercetin (Qu) through Src/Stat3 signaling. We found that the protein levels of S100A7, phosphorylated Src (p-Src), and p-Stat3 were increased in A431-III cells. Flavonoids Lu and Qu reduce protein levels of p-Src, p-Stat3 and S100A7 in A431-III cells. Treatment of A431-III cells with Src inhibitor SU6656 and Stat3 inhibitor S3I-201 also reduced the protein levels of S100A7. Transactivation activity of 5'-upstream regions of was activated by Stat3 but was reduced by treatment with Lu, Qu, SU6656 and S3I-201. The treatment also reduced the migratory and invasive abilities of A431-III cells. In a further analysis of EMT markers, the protein level of E-cad increased and that of Twist decreased after treatment with the inhibitors and flavonoids. Overexpression of S100A7 decreased the protein level of E-cad and increased the Twist level, whereas knockdown of S100A7 had the opposite effects. Treatment with S3I-201, Lu and Qu, compared to the control, were found to decrease metastasis of tumor cells in zebrafish larvae. These results suggest that Lu and Qu may inhibit Src/Stat3/S100A7 signaling to reduce tumorigenesis of cancer cells.
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http://dx.doi.org/10.3390/antiox8110557DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6912538PMC
November 2019

Structure of an antibiotic-synthesizing UDP-glucuronate 4-epimerase MoeE5 in complex with substrate.

Biochem Biophys Res Commun 2020 01 23;521(1):31-36. Epub 2019 Oct 23.

University of Chinese Academy of Sciences, Beijing, 100049, China; Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin, 300308, China; State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, China. Electronic address:

The epimerase MoeE5 from Streptomyces viridosporus converts UDP-glucuronic acid (UDP-GlcA) to UDP-galacturonic acid (UDP-GalA) to provide the first sugar in synthesizing moenomycin, a potent inhibitor against bacterial peptidoglycan glycosyltransferases. The enzyme belongs to the UDP-hexose 4-epimerase family, and uses NAD as its cofactor. Here we present the complex crystal structures of MoeE5/NAD/UDP-GlcA and MoeE5/NAD/UDP-glucose, determined at 1.48 Å and 1.66 Å resolution. The cofactor NAD is bound to the N-terminal Rossmann-fold domain and the substrate is bound to the smaller C-terminal domain. In both crystals the C4 atom of the sugar moiety of the substrate is in close proximity to the C4 atom of the nicotinamide of NAD, and the O4 atom of the sugar is also hydrogen bonded to the side chain of Tyr154, suggesting a productive binding mode. As the first complex structure of this protein family with a bound UDP-GlcA in the active site, it shows an extensive hydrogen-bond network between the enzyme and the substrate. We further built a model with the product UDP-GalA, and found that the unique Arg192 of MoeE5 might play an important role in the catalytic pathway. Consequently, MoeE5 is likely a specific epimerase for UDP-GlcA to UDP-GalA conversion, rather than a promiscuous enzyme as some other family members.
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http://dx.doi.org/10.1016/j.bbrc.2019.10.035DOI Listing
January 2020

Structural insights into the interaction between phytoplasmal effector causing phyllody 1 and MADS transcription factors.

Plant J 2019 11 9;100(4):706-719. Epub 2019 Sep 9.

The PhD Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 11529, Taiwan.

Phytoplasmas are bacterial plant pathogens which can induce severe symptoms including dwarfism, phyllody and virescence in an infected plant. Because phytoplasmas infect many important crops such as peanut and papaya they have caused serious agricultural losses. The phytoplasmal effector causing phyllody 1 (PHYL1) is an important phytoplasmal pathogenic factor which affects the biological function of MADS transcription factors by interacting with their K (keratin-like) domain, thus resulting in abnormal plant developments such as phyllody. Until now, lack of information on the structure of PHYL1 has prevented a detailed understanding of the binding mechanism between PHYL1 and the MADS transcription factors. Here, we present the crystal structure of PHYL1 from peanut witches'-broom phytoplasma (PHYL1 ). This protein was found to fold into a unique α-helical hairpin with exposed hydrophobic residues on its surface that may play an important role in its biological function. Using proteomics approaches, we propose a binding mode of PHYL1 with the K domain of the MADS transcription factor SEPALLATA3 (SEP3_K) and identify the residues of PHYL1 that are important for this interaction. Furthermore, using surface plasmon resonance we measure the binding strength of PHYL1 proteins to SEP3_K. Lastly, based on confocal images, we found that α-helix 2 of PHYL1 plays an important role in PHYL1-mediated degradation of SEP3. Taken together, these results provide a structural understanding of the specific binding mechanism between PHYL1 and SEP3_K.
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http://dx.doi.org/10.1111/tpj.14463DOI Listing
November 2019

Structural insights to heterodimeric cis-prenyltransferases through yeast dehydrodolichyl diphosphate synthase subunit Nus1.

Biochem Biophys Res Commun 2019 08 6;515(4):621-626. Epub 2019 Jun 6.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan, 430062, China. Electronic address:

The polyprenoid glycan carriers are produced by cis-prenyltransferases (cis-PTs), which function as heterodimers in metazoa and fungi or homodimers in bacteria, but both are found in plants, protista and archaea. Heterodimeric cis-PTs comprise catalytic and non-catalytic subunits while homodimeric enzymes contain two catalytic subunits. The non-catalytic subunits of cis-PT shows low sequence similarity to known cis-PTs and their structure information is of great interests. Here we report the crystal structure of Nus1, the non-catalytic subunit of cis-PT from Saccharomyces cerevisiae. We also investigate the heterodimer formation and active site conformation by constructing a homology model of Nus1 and its catalytic subunit. Nus1 does not contain an active site, but its C-terminus may participate in catalysis by interacting with the substrates bound to the catalytic subunit. These results provide important basis for further investigation of heterodimeric cis-PTs.
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http://dx.doi.org/10.1016/j.bbrc.2019.05.135DOI Listing
August 2019

Functional and structural investigations of fibronectin-binding protein Apa from Mycobacterium tuberculosis.

Biochim Biophys Acta Gen Subj 2019 09 5;1863(9):1351-1359. Epub 2019 Jun 5.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 430062, China. Electronic address:

Background: Alanine and proline-rich protein (Apa) is a secreted antigen of Mycobacterium spp. which involves in stimulating immune responses and adhering to host cells by binding to fibronectin (Fn). Here, we report the crystal structure of Apa from Mycobacterium tuberculosis (Mtb) and its Fn-binding characteristics.

Methods: The crystal structure of Mtb Apa was determined at resolutions of 1.54 Å. The dissociation constants (K) of Apa and individual modules of Fn were determined by surface plasmon resonance and enzyme-linked immunosorbent assay. Site-directed mutagenesis was performed to investigate the putative Fn-binding motif of Apa.

Results: Mtb Apa folds into a large seven-stranded anti-parallel β-sheet which is flanked by three α-helices. The binding affinity of Mtb Apa to individual Fn modules was assessed and the results indicated that the Mtb Apa binds to FnIII-4 and FnIII-5 of Fn CBD segment. Notably, structure analysis suggested that the previously proposed Fn-binding motif RWFV is buried within the protein and may not be accessible to the binding counterpart.

Conclusions: The structural and Fn-binding characteristics we reported here provide molecular insights into the multifunctional protein Mtb Apa. FnIII-4 and FnIII-5 of CBD are the only two modules contributing to Apa-Fn interaction.

General Significance: This is the first study to report the structure and Fn-binding characteristics of mycobacterial Apa. Since Apa plays a central role in stimulating immune responses and host cells adhesion, these results are of great importance in understanding the pathogenesis of mycobacterium. This information shall provide a guidance for the development of anti-mycobacteria regimen.
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http://dx.doi.org/10.1016/j.bbagen.2019.06.003DOI Listing
September 2019

Human DNA Polymerase μ Can Use a Noncanonical Mechanism for Multiple Mn-Mediated Functions.

J Am Chem Soc 2019 05 17;141(21):8489-8502. Epub 2019 May 17.

Institute of Biological Chemistry, Academia Sinica , 128 Academia Road Sec. 2 , Nankang, Taipei 115 , Taiwan.

Recent research on the structure and mechanism of DNA polymerases has continued to generate fundamentally important features, including a noncanonical pathway involving "prebinding" of metal-bound dNTP (MdNTP) in the absence of DNA. While this noncanonical mechanism was shown to be a possible subset for African swine fever DNA polymerase X (Pol X) and human Pol λ, it remains unknown whether it could be the primary pathway for a DNA polymerase. Pol μ is a unique member of the X-family with multiple functions and with unusual Mn preference. Here we report that Pol μ not only prebinds MdNTP in a catalytically active conformation but also exerts a Mn over Mg preference at this early stage of catalysis, for various functions: incorporation of dNTP into a single nucleotide gapped DNA, incorporation of rNTP in the nonhomologous end joining (NHEJ) repair, incorporation of dNTP to an ssDNA, and incorporation of an 8-oxo-dGTP opposite template dA (mismatched) or dC (matched). The structural basis of this noncanonical mechanism and Mn over Mg preference in these functions was analyzed by solving 19 structures of prebinding binary complexes, precatalytic ternary complexes, and product complexes. The results suggest that the noncanonical pathway is functionally relevant for the multiple functions of Pol μ. Overall, this work provides the structural and mechanistic basis for the long-standing puzzle in the Mn preference of Pol μ and expands the landscape of the possible mechanisms of DNA polymerases to include both mechanistic pathways.
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http://dx.doi.org/10.1021/jacs.9b01741DOI Listing
May 2019

Structural Insights to the Heterotetrameric Interaction between the PirA and PirB Toxins and Activation of the Cry-Like Pore-Forming Domain.

Toxins (Basel) 2019 04 22;11(4). Epub 2019 Apr 22.

The Ph.D. Program for Translational Medicine, College of Medical Science and Technology, Taipei Medical University and Academia Sinica, Taipei 110, Taiwan.

Acute hepatopancreatic necrosis disease (AHPND) is a newly emergent penaeid shrimp disease which can cause 70-100% mortality in and , and has resulted in enormous economic losses since its appearance. AHPND is caused by the specific strains of that harbor the pVA1 plasmid and express PirA and PirB toxins. These two toxins have been reported to form a binary complex. When both are present, they lead to the death of shrimp epithelial cells in the hepatopancreas and cause the typical histological symptoms of AHPND. However, the binding mode of PirA and PirB has not yet been determined. Here, we used isothermal titration calorimetry (ITC) to measure the binding affinity of PirA and PirB. Since the dissociation constant ( = 7.33 ± 1.20 μM) was considered too low to form a sufficiently stable complex for X-ray crystallographic analysis, we used alternative methods to investigate PirA-PirB interaction, first by using gel filtration to evaluate the molecular weight of the PirA/PirB complex, and then by using cross-linking and hydrogen-deuterium exchange (HDX) mass spectrometry to further understand the interaction interface between PirA and PirB. Based on these results, we propose a heterotetrameric interaction model of this binary toxin complex. This model provides insight of how conformational changes might activate the PirB N-terminal pore-forming domain and should be helpful for devising effective anti-AHPND strategies in the future.
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http://dx.doi.org/10.3390/toxins11040233DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6520838PMC
April 2019

Substrate-analogue complex structure of Mycobacterium tuberculosis decaprenyl diphosphate synthase.

Acta Crystallogr F Struct Biol Commun 2019 Apr 13;75(Pt 4):212-216. Epub 2019 Mar 13.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, School of Life Sciences, Hubei University, Wuhan 43420, People's Republic of China.

Decaprenyl diphosphate synthase from Mycobacterium tuberculosis (MtDPPS, also known as Rv2361c) catalyzes the consecutive elongation of ω,E,Z-farnesyl diphosphate (EZ-FPP) by seven isoprene units by forming new cis double bonds. The protein folds into a butterfly-like homodimer like most other cis-type prenyltransferases. The starting allylic substrate EZ-FPP is bound to the S1 site and the homoallylic substrate to be incorporated, isopentenyl diphosphate, is bound to the S2 site. Here, a 1.55 Å resolution structure of MtDPPS in complex with the substrate analogues geranyl S-thiodiphosphate (GSPP) and isopentenyl S-thiodiphosphate bound to their respective sites in one subunit clearly shows the active-site configuration and the magnesium-coordinated geometry for catalysis. The ligand-binding mode of GSPP in the other subunit indicates a possible pathway of product translocation from the S2 site to the S1 site, as required for the next step of the reaction. The preferred binding of negatively charged effectors to the S1 site also suggests a promising direction for inhibitor design.
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http://dx.doi.org/10.1107/S2053230X19001213DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6450523PMC
April 2019

Use of Cryo-EM To Uncover Structural Bases of pH Effect and Cofactor Bispecificity of Ketol-Acid Reductoisomerase.

J Am Chem Soc 2019 04 2;141(15):6136-6140. Epub 2019 Apr 2.

Institute of Biochemical Sciences , National Taiwan University , Taipei 106 , Taiwan.

While cryo-EM is revolutionizing structural biology, its impact on enzymology is yet to be fully demonstrated. The ketol-acid reductoisomerase (KARI) catalyzes conversion of (2 S)-acetolactate or (2 S)-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate. We found that KARI from archaea Sulfolobus solfataricus (Sso-KARI) is unusual in being a dodecamer, bispecific to NADH and NADPH, and losing activity above pH 7.8. While crystals were obtainable only at pH 8.5, cryo-EM structures were solved at pH 7.5 and 8.5 for Sso-KARI:2Mg. The results showed that the distances of the two catalytic Mg ions are lengthened in both structures at pH 8.5. We next solved cryo-EM structures of two Sso-KARI complexes, with NADH+inhibitor and NADPH+inhibitor at pH 7.5, which indicate that the bispecificity can be attributed to a unique asparagine at the cofactor binding loop. Unexpectedly, Sso-KARI also differs from other KARI enzymes in lacking "induced-fit", reflecting structural rigidity. Thus, cryo-EM is powerful for structural and mechanistic enzymology.
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http://dx.doi.org/10.1021/jacs.9b01354DOI Listing
April 2019

Reduction in MnSOD promotes the migration and invasion of squamous carcinoma cells.

Int J Oncol 2019 May 14;54(5):1639-1650. Epub 2019 Mar 14.

Department of Biochemistry and Molecular Cell Biology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 11031, Taiwan, R.O.C.

Reactive oxygen species (ROS) homeostasis is maintained at a higher level in cancer cells, which promotes tumorigenesis. Oxidative stress induced by anticancer drugs may further increase ROS to promote apoptosis, but can also enhance the metastasis of cancer cells. The effects of ROS homeostasis on cancer cells remain to be fully elucidated. In the present study, the effect of a reduction in manganese superoxide dismutase (MnSOD) on the migration and invasion of A431 cells was investigated. Our previous micro‑assay data revealed that the mRNA expression of MnSOD was higher in the invasive A431‑III cell line compared with that in the parental A431 cell line (A431‑P). In the present study, high protein levels of MnSOD and H2O2 production were observed in A431‑III cells; however, catalase protein levels were significantly lower in A431‑III cells compared with those in the A431‑P cell line. The knockdown of MnSOD increased H2O2 levels, enzyme activity, the mRNA levels of matrix metalloproteinase‑1, ‑2 and ‑9, and the migratory and invasive abilities of the cells. Inducing a reduction in H2O2 using diphenyleneiodonium (DPI) and N‑acetyl‑l‑cysteine decreased the migratory abilities of the cell lines, and DPI attenuated the migratory ability that had been increased by MnSOD small interfering RNA knockdown. Luteolin (Lu) and quercetin (Qu) increased the expression of catalase and reduced H2O2 levels, but without an observed change in the protein levels of MnSOD. Taken together, these data suggest that reduced MnSOD may induce ROS imbalance in cells and promote the metastatic ability of cancer cells. Lu and Qu may attenuate these processes and may be promising potential anticancer agents.
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http://dx.doi.org/10.3892/ijo.2019.4750DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6438424PMC
May 2019

An Effective Neutralizing Antibody Against Influenza Virus H1N1 from Human B Cells.

Sci Rep 2019 03 14;9(1):4546. Epub 2019 Mar 14.

Institute of Biological Chemistry, Academia Sinica, Taipei, Taiwan.

Influenza is a contagious acute respiratory disease caused by the influenza virus infection. Hemagglutinin (HA) is an important target in the therapeutic treatment and diagnostic detection of the influenza virus. Influenza A virus encompasses several different HA subtypes with different strains, which are constantly changing. In this study, we identified a fully human H1N1 neutralizing antibody (32D6) via an Epstein-Barr virus-immortalized B cell-based technology. 32D6 specifically neutralizes the clinically isolated H1N1 strains after the 2009 pandemic but not the earlier strains. The epitope was identified through X-ray crystallographic analysis of the 32D6-Fab/HA1 complex structure, which revealed a unique loop conformation located on the top surface of HA. The major region is composed of two peptide segments (residues 172-177 and 206-213), which form an abreast loop conformation. The residue T262 between the two loops forms a conformational epitope for recognition by 32D6. Three water molecules were observed at the interface of HA and the heavy chain, and they may constitute a stabilizing element for the 32D6-HA association. In addition, each 32D6-Fab is likely capable of blocking one HA trimer. This study provides important information on the strain specificity of 32D6 for the therapeutic treatment and detection of viral infection.
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http://dx.doi.org/10.1038/s41598-019-40937-4DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6418199PMC
March 2019

Complex structures of MoeN5 with substrate analogues suggest sequential catalytic mechanism.

Biochem Biophys Res Commun 2019 04 2;511(4):800-805. Epub 2019 Mar 2.

State Key Laboratory of Biocatalysis and Enzyme Engineering, School of Life Sciences, Hubei University, Wuhan, 4342008, China. Electronic address:

The antibiotic moenomycin A is a phosphoglycerate derivative with a C-moenocinyl chain and a branched oligosaccharide. Formation of the C-chain is catalyzed by the enzyme MoeN5 with geranyl pyrophosphate (GPP) and the sugar-linked 2-Z,E-farnesyl-3-phosphoglycerate (FPG) as its substrates. Previous complex crystal structures with GPP and long-chain alkyl glycosides suggested that GPP binds to the S1 site in a similar way as in most other α-helical prenyltransferases (PTs), and FPG is likely to assume a bent conformation in the S2 site. However, two FPG derivatives synthesized in the current study were found in the S1 site rather than S2 in their complex crystal structures with MoeN5. Apparently S1 is the preferred site for prenyl-containing ligand, and S2 binding may proceed only after S1 is occupied. Thus, like most trans-type PTs, MoeN5 may employ a sequential ionization-condensation-elimination mechanism that involves a carbocation intermediate.
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http://dx.doi.org/10.1016/j.bbrc.2019.02.131DOI Listing
April 2019

Crystal structure of the blue fluorescent protein with a Leu-Leu-Gly tri-peptide chromophore derived from the purple chromoprotein of Stichodactyla haddoni.

Int J Biol Macromol 2019 Jun 2;130:675-684. Epub 2019 Mar 2.

Institute of Biomedical Sciences, MacKay Medical College, New Taipei City, Taiwan. Electronic address:

Chromoproteins are a good source of engineered biological tools. We previously reported the development of a blue fluorescent protein, termed shBFP, which was derived from a purple chromoprotein shCP found in the sea anemone Stichodacyla haddoni. shBFP contains a Leu-Leu-Gly tri-peptide chromophore, and shows maximum excitation and emission wavelengths at 401 nm and 458 nm, along with a high quantum yield. How this chromophore endows shBFP with the unique fluorescence property in the absence of a hydroxyphenyl ring remained unclear. Here, we present the crystal structures of shCP and shBFP at 1.9- and 2.05-Å resolution, respectively. Both proteins crystallized as similar tetramers, but they are more likely to function as dimers in solution. The chromophore in shCP shows a trans-conformation and its non-planarity is similar to most other homologues. The shBFP chromophore also contains an imidazolidone moiety in its structure, but there are a smaller number of conjugated double bonds compared to shCP. Consequently, the chromophore may prefer absorbing shorter wavelength lights in the UV region, followed by the emission of blue fluorescence. These observations provide new insights into the molecular basis that correlates chromophore conformation with light absorption and fluorescence emission for the development of improved biomarkers.
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http://dx.doi.org/10.1016/j.ijbiomac.2019.02.138DOI Listing
June 2019

Crystal structure of LepI, a multifunctional SAM-dependent enzyme which catalyzes pericyclic reactions in leporin biosynthesis.

Org Biomol Chem 2019 02;17(8):2070-2076

Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.

LepI is a novel multifunctional enzyme that catalyzes stereoselective dehydration, Diels-Alder reaction, and retro-Claisen rearrangement. Here we report the crystal structure of LepI in complex with its co-factor S-adenosyl methionine (SAM). LepI forms a tetramer via the N-terminal helical domain and binds to a SAM molecule in the C-terminal catalytic domain. The binding modes of various LepI substrates are investigated by docking simulations, which suggest that the substrates are bound via both hydrophobic and hydrophilic forces, as well as cation-π interactions with the positively charged SAM. The reaction starts with a dehydration step in which H133 possibly deprotonates the pyridone hydroxyl group of the substrate, while D296 might protonate an alkyl-chain hydroxyl group. Subsequent pericyclization may be facilitated by the correct fold of the substrate's alkyl chain and a thermodynamic driving force towards σ-bonds at the expense of π-bonds. These results provide structural insights into LepI catalysis and are important in understanding the mechanism of enzymatic pericyclization.
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http://dx.doi.org/10.1039/c8ob02758gDOI Listing
February 2019

Structure of undecaprenyl pyrophosphate synthase from Acinetobacter baumannii.

Acta Crystallogr F Struct Biol Commun 2018 Dec 16;74(Pt 12):765-769. Epub 2018 Nov 16.

Department of Biotechnology, HungKuang University, Taichung, Taiwan.

Undecaprenyl pyrophosphate (UPP) is an important carrier of the oligosaccharide component in peptidoglycan synthesis. Inhibition of UPP synthase (UPPS) may be an effective strategy in combating the pathogen Acinetobacter baumannii, which has evolved to be multidrug-resistant. Here, A. baumannii UPPS (AbUPPS) was cloned, expressed, purified and crystallized, and its structure was determined by X-ray diffraction. Each chain of the dimeric protein folds into a central β-sheet with several surrounding α-helices, including one at the C-terminus. In the active site, two molecules of citrate interact with the side chains of the catalytic aspartate and serine. These observations may provide a structural basis for inhibitor design against AbUPPS.
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http://dx.doi.org/10.1107/S2053230X18012931DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6277960PMC
December 2018

Catalytic Role of Conserved Asparagine, Glutamine, Serine, and Tyrosine Residues in Isoprenoid Biosynthesis Enzymes.

ACS Catal 2018 May 6;8(5):4299-4312. Epub 2018 Apr 6.

Industrial Enzymes National Engineering Laboratory, Tianjin Institute of Industrial Biotechnology, Chinese Academy of Sciences, Tianjin 300308, China.

We report the results of an investigation into the catalytic role of highly conserved amide (asparagine, glutamine) and OH-containing (serine, tyrosine) residues in several prenyltransferases. We first obtained the X-ray structure of cyclolavandulyl diphosphate synthase containing two molecules of the substrate analog dimethylallyl ()-thiolodiphosphate (DMASPP). The two molecules have similar diphosphate group orientations to those seen in other ζ-fold (- head-to-tail and head-to-middle) prenyltransferases with one diphosphate moiety forming a bidentate chelate with Mg in the so-called S1 site (which is typically the allylic binding site in ζ-fold proteins) while the second diphosphate binds to Mg in the so-called S2 site (which is typically the homoallylic binding site in ζ-fold proteins) a single P1O1 oxygen. The latter interaction can facilitate direct phosphate-mediated proton abstraction P1O2, or more likely by an indirect mechanism in which P1O2 stabilizes a basic asparagine species that removes H, which is then eliminated an Asn-Ser shuttle. The universal occurrence of Asn-Ser pairs in ζ-fold proteins leads to the idea that the highly conserved amide (Asn, Gln) and OH-containing (Tyr) residues seen in many "head-to-head" prenyltransferases such as squalene and dehydrosqualene synthase might play similar roles, in H elimination. Structural, bioinformatics and mutagenesis investigations indeed indicate an important role of these residues in catalysis, with the results of density functional theory calculations showing that Asn bound to Mg can act as a general (imine-like) base, while Gln, Tyr and HO form a proton channel that is adjacent to the conventional (Asp-rich) "active site". Taken together, our results lead to mechanisms of proton-elimination from carbocations in numerous prenyltransferases in which neutral species (Asn, Gln, Ser, Tyr, HO) act as proton shuttles, complementing the more familiar roles of acidic groups (in Asp and Glu) that bind to Mg, and basic groups (primarily Arg) that bind to diphosphates, in isoprenoid biosynthesis.
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http://dx.doi.org/10.1021/acscatal.8b00543DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6193494PMC
May 2018

The Crystal Structure of a Class of Cyclases that Catalyze the Cope Rearrangement.

Angew Chem Int Ed Engl 2018 11 23;57(46):15060-15064. Epub 2018 Oct 23.

State Key Laboratory of Biocatalysis and Enzyme Engineering, Hubei Collaborative Innovation Center for Green Transformation of Bio-resources, Environmental Microbial Technology Center of Hubei Province, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, 430062, China.

Found recently in stignomatales, the Stig cyclases catalyze the Cope rearrangement and intramolecular cyclization to produce complex indole alkaloids. Five crystal structures were solved of subfamily 1 and 2 Stig cyclases, which adopt a β-sandwich fold like the non-catalytic carbohydrate-binding motif. Several complex structures were also determined of indole-based compounds, which are bound to the hydrophobic terminal cavity, where a conserved Asp residue makes an H-bond to the indole N and triggers the acid-catalyzed Cope rearrangement. Through analyzing the enzyme-ligand interactions and mutagenesis experiments, several aromatic residues were found important in catalysis. Apart from a common substrate binding mode and catalytic mechanism, potential subfamily variations that may attribute to the different product specificity are implicated. These results shall expand our scope of enzymology, in particular for further investigation of the biosynthetic Cope rearrangement.
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http://dx.doi.org/10.1002/anie.201808231DOI Listing
November 2018

Crystal Structure of PigA: A Prolyl Thioester-Oxidizing Enzyme in Prodigiosin Biosynthesis.

Chembiochem 2019 01 11;20(2):193-202. Epub 2018 Oct 11.

Institute of Biological Chemistry, Academia Sinica, 128 Academia Road Section 2, Taipei, 11529, Taiwan.

Prodigiosin is an intensely red pigment comprising three pyrroles. The biosynthetic pathway includes a two-step proline oxidation catalyzed by phosphatidylinositol N-acetylglucosaminyltransferase subunit A (PigA), with flavin adenine dinucleotide (FAD) as its cofactor. The enzyme is crystallized in the apo form and in complex with FAD and proline. As an acyl coenzyme A dehydrogenase (ACAD) family member, the protein folds into a β-sheet flanked by two α-helical domains. PigA forms a tetramer, which is consistent with analytical ultracentrifugation results. FAD binds to PigA in a similar way to that in the other enzymes of the ACAD family. The variable conformations of loop β4-β5 and helix αG correlate well with the structural flexibility required for substrate entrance to the Re side of FAD. Modeling with PigG, the acyl carrier protein, suggests a reasonable mode of interaction with PigA. The structure helps to explain the proline oxidation mechanism, in which Glu244 plays a central role by abstracting the substrate protons. It also reveals a plausible pocket for oxygen binding to the Si side of FAD.
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http://dx.doi.org/10.1002/cbic.201800409DOI Listing
January 2019

Structural studies reveal the molecular mechanism of PETase.

FEBS J 2018 10 17;285(20):3717-3723. Epub 2018 Aug 17.

Hubei Collaborative Innovation Center for Green Transformation of Bio-Resources, Hubei Key Laboratory of Industrial Biotechnology, College of Life Sciences, Hubei University, Wuhan, China.

Poly(ethylene terephthalate) (PET) is a class of plastic material widely used in modern society, but large amounts of PET waste cause severe environmental problems. Obtained from a PET-consuming bacterium Ideonella sakaiensis, the enzyme PETase exhibits superb hydrolytic activity and substrate preference toward PET. Here, we summarize some recent advances in the crystallographic analysis of PETase. These reports uncover structural features of PETase that are involved in its catalytic activity. In comparison to homologous enzymes, PETase contains an additional disulfide bond as well as an extended β8-α6 loop. More importantly, the crystal structures of PETase in complex with substrate and product analogs provide critical information for understanding the mechanism of action of PETase. In particular, the wobbling conformation of W156 is closely related to the binding of substrate and product. These new findings are of great importance for further in-depth research and engineering development of PETase, and should advance the implementation of plastic biodegradation strategy.
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http://dx.doi.org/10.1111/febs.14612DOI Listing
October 2018