Education
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Postdoctoral fellow, University of Texas Southwestern Medical Center at Dallas
Subjects currently investigated and selected results
Function characterization of the replication protein of Bamboo mosaic virus
Bamboo mosaic virus (BaMV) is a positive-stranded RNA virus belonging to the genus Potexvirus. The 6.4-kb genome contains five open reading frames (ORFs) with a 5’ methyl cap and a 3’ poly(A) tail. A serial of biochemical studies have demonstrated that ORF1 of BaMV encodes a 155-kDa replication protein (REP) which can be functionally divided into an N-terminal mRNA capping enzyme, a helicase-like RNA 5’-triphosphatase, and a C-terminal RNA-dependent RNA polymerase. The capping enzyme exhibits a novel S-adenosylmethionine (AdoMet)-dependent guanylyltransferase activity, which can be dissected into GTP methyltransferase and m7GTP: mRNA guanylyltransferase (Figure 1). To form the 5’ methyl cap, GTP is first methylated on the N-7 of guanine by GTP methyltransferase using AdoMet as the methyl group donor, and the m7GMP moiety of the m7GTP is then transferred by m7GTP: mRNA guanylyltransferase to the 5’-diphosphated mRNA. In detail, m7GTP is first attacked by the nucleophilic imidazole of the active-site histidine (H68), leading to the formation of a covalent [Enzyme-m7GMP] intermediate, and m7GMP on the intermediate is next transferred to the 5’-diphosphated end of mRNA. Similar activities have also been observed on the capping enzymes of Semlike Forest virus, Brome mosaic virus, and Tobacco mosaic virus (all are members of alphavirus-like superfamily); therefore, all the viruses of alphavirus-like superfamily are believed to possess such a novel cap-formation mechanism.
The RNA triphosphatase domain of BaMV removes the gamma phosphate of nascent mRNA, generating a 5’-diphosphated end ready to accept m7GMP from the covalent [Enzyme-m7GMP] intermediate. Since the enzyme contains featured motifs of helicase, it exemplifies a new family of RNA 5’-triphosphatase, different from those of metazoan and plant that are classified into a metal-independent cysteine phosphatase superfamily, and of fungi, protozoa, and certain DNA viruses that are grouped as a metal-dependent phosphohydrolase superfamily. Besides, the viral triphosphatase is able to bind the viral coat protein. Reducing the binding strength between the two proteins restricts BaMV from cell-to-cell movement. Accordingly, we believe that the movement entity of BaMV contains the replication protein so that the virus can establish its replication territory soon after it enters new host cells. The RNA-dependent RNA polymerase of BaMV specifically recognizes the 5’- and 3’-untranslated regions of the viral genome, and it is responsible for the viral genome replication.
Currently, a great effort is put into the isolation of the membrane-bound viral replication protein complex, from which host factors potentially involved in the viral replication function are identified by LC-tandem Mass spectrometry. The effects of the factors on BaMV replication are being investigated.
The RNA triphosphatase domain of BaMV removes the gamma phosphate of nascent mRNA, generating a 5’-diphosphated end ready to accept m7GMP from the covalent [Enzyme-m7GMP] intermediate. Since the enzyme contains featured motifs of helicase, it exemplifies a new family of RNA 5’-triphosphatase, different from those of metazoan and plant that are classified into a metal-independent cysteine phosphatase superfamily, and of fungi, protozoa, and certain DNA viruses that are grouped as a metal-dependent phosphohydrolase superfamily. Besides, the viral triphosphatase is able to bind the viral coat protein. Reducing the binding strength between the two proteins restricts BaMV from cell-to-cell movement. Accordingly, we believe that the movement entity of BaMV contains the replication protein so that the virus can establish its replication territory soon after it enters new host cells. The RNA-dependent RNA polymerase of BaMV specifically recognizes the 5’- and 3’-untranslated regions of the viral genome, and it is responsible for the viral genome replication.
Currently, a great effort is put into the isolation of the membrane-bound viral replication protein complex, from which host factors potentially involved in the viral replication function are identified by LC-tandem Mass spectrometry. The effects of the factors on BaMV replication are being investigated.
Bacterial lignin-degrading polyphenol oxidas
Agricultural lignocelluloses could represent great resources for the production of biofuels and renewable chemicals if the recalcitrant lignin polymer could be efficiently removed without negatively impacting the environment. Recently, a monocopper polyphenol oxidase (Tfu1114) from Thermobifida fusca was found capable of assisting the bacterium’s own xylanase/cellulase to release reducing sugars from sugarcane bagasse. In Tfu1114-catalyzed reaction, molecular oxygen is reduced to hydrogen peroxide, coupled to the oxidation of 2,6-dimethoxyphenol, alkaline lignin, or sugarcane bagasse. Tfu1114 can reduce the total phenolic content of alkaline lignin and shift the molecular weight distribution to species with smaller sizes. Dilignol subunits such as syringaresinol and simulanol are generated in the hydrolysate of bagasse treated with Tfu1114. FTIR analysis suggests that Tfu1114 removed some C-C and/or C-O bonds adjacent to aromatic rings of lignin. From an applied perspective, Tfu1114 and cellulases acted in concert to release reducing sugars that are as usable as glucose for the cultivation of Escherichia coli, suggesting that toxins such as furfural and furan are absent, or at ignorable concentrations, in the hydrolysate. Tfu1114, therefore, represents a new type of lignin-degrading enzyme with potential to provide an eco-friendly way to modify the lignin in lignocellulose. The detail of Tfu1114 can be viewed in reports (Chen et al., 2016, Proc. Biochem. 51:1486-1495) and (Chen et al., 2013, Appl. Microbiol. Biotechnol. 97:8977-8986)
Engineering Rhodococcus equi USA-18 to produce steroid drug intermediates
R. equi USA-18, a non-pathogenic strain, grows robustly in media containing sterols and secrets a great amount of cholesterol oxidase. Recently, the bacterial gene encoding the reductase component (KshB) of 3-ketosteroid 9a-hydroxylase was deleted by a PCR-targeted gene disruption method and the change in sterol catabolism in response to the deletion was investigated. The knockout strain accumulates 3-oxo-23,24-bisnorchola-1,4-dien-22-oic acid and 1,4-androstadiene-3,17-dione, both important intermediates for steroid drug synthesis, in the culture medium that contains cholesterol or phytosterols. More genetic tools for the engineering of strains of R. equi are being developed so that mutant strains with modified steroid catabolic pathways can be created.
The structure-function relationship of phosphoglucose isomerase
Phosphoglucose isomerase (PGI) is a protein with multiple functions. As being a glycolytic enzyme, it catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate in the glycolysis and gluconeogenesis pathways. Outside the cells, PGI from mammals also function as neuroleukin supporting the growth of nerve cells, autocrine motility factor of tumor cells, differentiation and maturation mediator for myeloid leukemia cells, and autoantigen able to trigger rheumatoid arthritis. To understand the structure of the protein and its catalytic mechanism for converting phosphosugars, the enzyme originally from Bacillus stearothermophilus was purified and crystallized with or without the presence of substrate analogs. The 3-D structures indicate that the enzyme is a dimmer. Each subunit is composed of a large and a small globular domain with similar structure in which a beta-sheet core is surrounded by alpha-helices. The active site is located within the two globular domains and the interface between the two subunits. Differential effects of N-bromo-acetylethanolamine phosphate, an active-site directed inhibitor, on the wild-type and mutant enzymes suggest a base role for His306, the sole active-site histidine, in the isomerization reaction. Site-directed mutagenesis also suggests functions for each of the conserved cationic and anionic amino acid residues surrounding the active site. Combing with the structure information, we proposed a mechanism for the phosphoglucose isomeras-catalyzed isomerization (Figure 2). In brief, His306 and Glu285 work as a general base-acid pair to prompt the formation of cis-enediol intermediate. By reversing their roles in the subsequent step, the intermediate is then transformed to the product. Through electrostatic interaction, Glu145 could stabilize the partial positive charge developing on the side chain of His306 during the isomerization step. Glu145 also forms a hydrogen bond to His306 and may lock the imidazole ring of His306 in an optimal position for reaction. For details, please refer to Chou et al. (2000) J Bio Chem 275:23154-23160.
GPI deficiency is the second most common erythroenzymopathy of glycolytic enzymes after pyruvate kinase deficiency. To gain a more complete understanding of the molecular basis for the nonspherocytic hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants had been expressed in Escherichia coli, purified to homogeneity, and functionally characterized. The mechanistic basis of the anemia caused by each of the variants can refer to the Lin et al (2009) Biochim Biophys Acta (Proteins and Proteomics) 1794:315-323.
GPI deficiency is the second most common erythroenzymopathy of glycolytic enzymes after pyruvate kinase deficiency. To gain a more complete understanding of the molecular basis for the nonspherocytic hemolytic anemia due to the GPI-deficiency, the wild-type enzyme and sixteen genetic variants had been expressed in Escherichia coli, purified to homogeneity, and functionally characterized. The mechanistic basis of the anemia caused by each of the variants can refer to the Lin et al (2009) Biochim Biophys Acta (Proteins and Proteomics) 1794:315-323.