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Honghai Wang

Publications and source records attributed to Honghai Wang.

18 recordsLinked to original sources
PubMed2006

Purification and characterization of a functionally active Mycobacterium tuberculosis prephenate dehydrogenase.

Tuberculosis (TB) remains to be a global health problem. New drugs are badly needed to drastically reduce treatment time and overcome some of the challenges with tuberculosis treatment, such as multi-drug resistant (MDR) strain infected patients or tuberculosis/HIV co-infected patients. The essentiality of mycobacterial aromatic amino acid biosynthesis pathways and their absence from human host indicate that the member enzymes of these pathways promising drug targets for therapeutic agents against pathogen mycobacteria. Prephenate dehydrogenase (PDH) is a key regulatory enzyme in tyrosine biosynthesis, catalyzing the NAD(+)-dependent conversion of prephenate to p-hydroxyphenylpyruvate, making it a potential drug target for antibiotics discovery. The recombinant PDH with an N-terminal His-tag (His-rMtPDH) was first purified in Escherichia coli, and using enterokinase rMtPDH was obtained by cleaving the N-terminal fusion partner. The effect of pH, temperature and the cation-Na(+) on purified enzyme activity was characterized. The N-terminal fusion partner was found to have little effect on the biochemical properties of PDH. We also provide in vitro evidence that Mycobacterium tuberculosis PDH does not possess any chorismate mutase (CM) activity, which suggests that, unlike many other enteric bacteria (where PDH exists as a fusion protein with CM), M. tuberculosis PDH is a monofunctional protein.

Anti-Infective Agents↗
PubMed2006

Characterization and site-directed mutagenesis of the putative novel acyl carrier protein Rv0033 and Rv1344 from Mycobacterium tuberculosis.

Mycolic acids are generated in Mycobacterium tuberculosis as a result of the interaction of two fatty acid biosynthetic systems: type I fatty acid synthase (FAS) and type II fatty acid synthase. Acyl carrier protein (ACP) is a small, acidic protein in type II FAS systems. It plays a central role in mycolic acid biosynthesis by transferring the acyl groups from one enzyme to another for the completion of the fatty acid synthesis cycle. The nature of the proper recognition between ACPs and its many interactive proteins is not understood. Here, we report the over-expression, purification, and characterization of two putative ACPs: Rv0033 and Rv1344 in M. tuberculosis. In order to study the role of the conserved residues and the conformation of whole protein, some site-directed mutations of recombinant Acp1344 were made and the 3D structure of Acp1344 was modeled.

Acyl Carrier Protein↗
PubMed2006

MSTF: a domain involved in bacterial metallopeptidases and surface proteins, mycobacteriophage tape-measure proteins and fungal proteins.

Here we report a novel domain, MSTF (domain involved in bacterial metallopeptidases, surface proteins and other proteins, also present in mycobacteriophage tape-measure proteins and fungal proteins), which is present in bacteria, phages and fungi. MSTF is about 67-94 amino acids in length with one HxDHxH motif and some highly conserved residues including His, Gly, Ala and Asp. Secondary structure prediction indicated that this domain contains two alpha-helices and one beta-sheet. Identification of MSTF will provide an opportunity to develop new strategies to combat pathogenic microorganisms, especially Mycobacterium tuberculosis.

Amino Acid Sequence↗
PubMed2006

Purification and characterization of Mycobacterium tuberculosis indole-3-glycerol phosphate synthase.

Indole-3-glycerol phosphate synthase (IGPS) plays an important role in the survival of Mycobacterium tuberculosis. The trpC gene, encoding IGPS, is essential for the growth of M. tuberculosis. It was expressed at the transcriptional level in cultured M. tuberculosis. The recombinant IGPS with an added His-tag was purified. The His-tag was found to have a small effect on the biochemical properties of IGPS. IGPS is a monofunctional enzyme in M. tuberculosis. Recombinant IGPS has considerable beta-pleated sheet and is relatively compact. The enzyme activity is significantly inhibited by denaturants and antibiotics, suggesting that IGPS may be a novel potential drug target of M. tuberculosis.

Bacterial Proteins↗
PubMed2005

Rv2131c gene product: an unconventional enzyme that is both inositol monophosphatase and fructose-1,6-bisphosphatase.

Inositol monophosphatase is an enzyme in the biosynthesis of myo-inostiol, a crucial substrate for the synthesis of phosphatidylinositol, which has been demonstrated to be an essential component of mycobacteria. In this study, the Rv2131c gene from Mycobacterium tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the enzyme in fusion with a histidine-rich peptide on the N-terminal. The fusion protein was purified from the soluble fraction of the lysed cells under native conditions by immobilized metal affinity chromatography (IMAC). The purified Rv2131c gene product showed inositol monophosphatase activity but with substrate specificity that was broader than those of several bacterial and eukaryotic inositol monophosphatases, and it also acted as fructose-1,6-bisphosphatase. The dimeric enzyme exhibited dual activities of IMPase and FBPase, with K(m) of 0.22+/-0.03mM for inositol-1-phosphate and K(m) of 0.45+/-0.05mM for fructose-1,6-bisphosphatase. To better understand the relationship between the function and structure of the Rv2131c enzyme, we constructed D40N, L71A, and D94N mutants and purified these corresponding proteins. Mutations of D40N and D94N caused the proteins to almost completely lose both the inositol monophosphatase and fructose-1,6-bisphosphatase activities. However, L71A mutant did not cause loss either of the activities, but the activity toward the inositol was 12-fold more resistant to inhibition by lithium (IC(50) approximately 60mM). Based on the substrate specificity and presence of conserved sequence motifs of the M. tuberculosis Rv2131c, we proposed that the enzyme belonged to class IV fructose-1,6-bisphosphatase (FBPase IV).

Amino Acid Sequence↗
PubMed2005

Expression, purification and properties of shikimate dehydrogenase from Mycobacterium tuberculosis.

Tuberculosis, caused by Mycobacterium tuberculosis, continues to be one of the main diseases to mankind. It is urgent to discover novel drug targets for appropriate antimicrobial agents against this human pathogen. The shikimate pathway is considered as an attractive target for the discovery of novel antibiotics for its essentiality in bacteria and absence in mammalian cells. The Mycobacterium tuberculosis aroE-encoded shikimate dehydrogenase was cloned, expressed and purified. Sequence alignment analysis shows that shikimate dehydrogenase of Mycobacterium tuberculosis exhibit the pattern of G-X-(N/S)-V-(T/S)-X-PX-K, which is highly conserved within the shikimate dehydrogenase family. The recombinant shikimate dehydrogenase spectrum determined by CD spectroscopy showed that the percentages for alpha-helix, beta-sheet, beta-turn, and random coil were 29.2 %, 9.3 %, 32.7 %, and 28.8 %, respectively. The enzymatic characterization demonstrates that it appears to be fully active at pH from 9.0 to 12, and temperature 63(o)C. The apparent Michaelis constant for shikimic acid and NADP(+) were calculated to be about 29.5 microM and 63 microM. The recombinant shikimate dehydrogenase catalyzes the substrate in the presence of NADP(+) with an enzyme turnover number of 399 s(-1). Zymological studies suggest that the cloned shikimate dehydrogenase from M. tuberculosis has a pretty activity, and the work should help in the discovery of enzyme inhibitors and further of possible antimicrobial agents against Mycobacterium tuberculosis.

Alcohol Oxidoreductases↗
PubMed2005

Purification and characterization of a functionally active Mycobacterium tuberculosis pyrroline-5-carboxylate reductase.

Pyrroline-5-carboxylate reductase (P5CR) plays an important role in the survival of Mycobacterium tuberculosis and is related to virulence of this pathogen. RT-PCR analysis indicated that proC, encoding P5CR, was expressed at the transcriptional level cultured in vitro. The His-rMtP5CR with an N-terminal His-tag (His-rMtP5CR) was firstly purified in Escherichia coli and rMtP5CR was obtained by removal of the N-terminal fusion partner using enterokinase. His-rMtP5CR had considerable beta-pleated sheet analyzed by circular dichroism spectroscopy. The effect of pH, temperature, cations, denaturants, and detergents on the purified enzyme activity and stability was characterized. The N-terminal fusion partner was found to have very little effect on the biochemical properties of P5CR.

Cells, Cultured↗
PubMed2005

Mesoporous silica nanoreactors for highly efficient proteolysis.

Protein digestion inside the nanoreactor channels of mesoporous silica (SBA-15) is reported, and evaluated by using peptide-mass mapping. Both proteases and substrates were efficiently captured within these biocompatible nanoreactors. After 10 minutes, the mass spectrum of the protein digests released from the mesoporous-silica-based nanoreactors revealed the presence of eight peptides covering 58% of the protein sequence with an intense signal (signal/noise ratio > 70). In comparison, the conventional overnight in-solution digestion of proteins under otherwise identical conditions generated only three peptides (27% sequence coverage). We propose that this order-of-magnitude increase in the proteolytic reaction rate is mainly attributed to two factors: substrate enrichment within mesoporous silica channels and enzyme immobilization. The surface properties and macrostructure of the mesoporous silica were studied to reveal their significant influence on proteolytic reactions.

Hydrolysis↗
PubMed2005

The Influence of Mycobacterium tuberculosis sigma factors on the promotion efficiency of ptpAt promoter in Mycobacterium smegmatis.

It was found in a previous study that Mycobacterium tuberculosis protein tyrosine phosphatase ptpAt promoter is a highly active promoter in slow-growing species of mycobacteria, such as M. tuberculosis and M. bovis BCG, but inert in fast-growing mycobacterial species, such as M. smegmatis. This difference is presumed to be due to the differences between sigma factors systems of slow-growing pathogenic mycobacteria and the fast-growing saprophyte M. smegmatis. Therefore, we constructed a series of plasmids, named pOLYG-13x, which can express various M. tuberculosis sigma factors and also contain a P(ptpAt)-gfp reporter gene construct. By inducing different sigma factor genes of M. tuberculosis in M. smegmatis, we were able to explore the influences of various sigma factors on the expression efficiency of the ptpAt promoter. The result show that of the 10 sigma factors evaluated, only sigF and sigL were able to weakly drive the ptpAt promoter in M. smegmatis and other sigma factors were unable to drive the promoter.

Bacterial Proteins↗
PubMed2005

Identification of a novel domain--DIM, which defines a new family composed mainly of bacterial membrane proteins.

We report here the identification of a novel domain - DIM (N-terminal domain in bacterial membrane proteins and other proteins) present exclusively in bacterial species including mycobacteria, revealed by PSI-BLAST iterative searches. DIM comprises about 53 amino acids in length with conserved Leu, Ile and Gly residues. Secondary structure prediction indicated that this domain contains two alpha-helices. DIM occurs at the N-terminus of proteins, and was found particularly but not exclusively in proteins with a transmembrane domain, and also in proteins with a FHA domain or RPT repeats. DIM-containing proteins have been reported to be involved in pathogenicity, signal transduction or small solute transport.

Amino Acid Sequence↗
PubMed2005

Expression and purification of a functionally active recombinant GDP-mannosyltransferase (PimA) from Mycobacterium tuberculosis H37Rv.

Lipoarabinomannans (LAM), especially mannose-capped LAM, abundant in the cell wall of Mycobacterium tuberculosis (Mtb) exhibit a broad spectrum of immunomodulatory functions and emerge as key virulence factors that may be relevant drug targets. The pimA gene of mycobacteria encodes a alpha-mannosyltransferase involved in the transfer reaction of the very first mannose from GDP-mannose to the carrier lipid phosphatidyl-myo-inositol, a precursor in the synthesis of LAM. PimA has been proposed to play an essential role in the growth of mycobacteria. In this study, the pimA gene from M. tuberculosis H37Rv was cloned into the pET28a vector and the recombinant plasmid was transformed into Escherichia coli BL21 (DE3) strain, allowing the expression of the Mtb PimA in fusion with a histidine-rich peptide on the N-terminal. The Mtb PimA was purified from the supernatant of the lysed cells under native conditions by immobilized metal affinity chromatography. The purity and molecular weight of Mtb PimA were determined by high performance liquid chromatography and matrix-assisted laser desorption ionization time-of-flight. Circular dichroism spectroscopy study on Mtb PimA showed that the protein was folded. The enzyme assays revealed that Mtb PimA showed a requirement for Mg(2+) for the activity and the K(m) and V(max) values of Mtb PimA were estimated at 18 +/- 2 microM and 0.1 +/- 0.05 nmol/min/microg, respectively. This is the first report describing cloning and expression of GDP-mannosyltransferase gene of M. tuberculosis in E. coli.

Bacterial Proteins↗
PubMed2004

Microfluidic enzymatic-reactors for peptide mapping: strategy, characterization, and performance.

The design and characterization of two kinds of poly(dimethylsiloxane)(PDMS) microfluidic enzymatic-reactors along with their analytical utility coupled to MALDI TOF and ESI MS were reported. Microfluidic devices integrated with microchannel and stainless steel tubing (SST) was fabricated using a PDMS casting technique, and was used for the preparation of the enzymatic-reactor. The chemical modification was performed by introducing carboxyl groups to PDMS surface based on ultraviolet graft polymerization of acrylic acid. The covalent and physical immobilization of trypsin was carried out with the use of the activation reagents 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide(EDC)/N-hydroxysuccinimide (NHS) and a coupling reagent poly(diallyldimethylammonium chloride)(PDDA), respectively. The properties and success of processes of trypsin immobilization were investigated by measuring contact angle, infrared absorption by attenuated total reflection spectra, AFM imaging and electropherograms. An innovative feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by embedded SST electrode and replaceable tip. The lab-made devices provide an excellent extent of digestion of several model proteins even at the fast flow rate of 3.5 microL min(-1) for the EDC/NHS-made device and 0.8 microL min(-1) for the PDDA-made device, which afford very short residence times of 5 s and 20 s, respectively. In addition, the lab-made devices are less susceptive to memory effect and can be used for at least 50 runs in one week without noticeable loss of activity. Moreover, the degraded PDDA-made device can be regenerated by simple treatment of a HCl solution. These features are the most required for microfluidic devices used for protein analysis.

Dimethylpolysiloxanes↗
PubMed2004

Detection of rifampin-resistant Mycobacterium tuberculosis strains by using a specialized oligonucleotide microarray.

DNA microarray represents one of the major advances in diagnostic sequencing of polymerase chain reaction (PCR) products. Until now, arrays have been relatively expensive, complex to perform, and difficult to interpret, limiting their wide application in the clinical laboratory. A moderate-density oligonucleotide microarray that can rapidly identify Mycobacterium tuberculosis rifampin-resistant strains was developed. The method is based on the detection of point mutations and other rearrangements in the rpoB gene region determining rifampin resistance. Rifampin resistance was determined by hybridizing fluorescently labeled, amplified genetic material generated from bacterial colonies to the array. Fifty-three rifampin-resistant M. tuberculosis and 15 rifampin-susceptible M. tuberculosis were tested and results were concordant with those based on culture drug susceptibility testing and sequencing. Rifampin-resistant clinical isolates were detected in as little as 1.5 hours after PCR amplification with visual results. It is demonstrated that oligonucleotide microarray is an efficient, specialized technique to implement and can be used as a rapid method for detecting rifampin resistance to complement standard culture-based method.

Antibiotics, Antitubercular↗
PubMed2003

Mutations in the rpoB gene of multidrug-resistant Mycobacterium tuberculosis isolates from China.

Mutations in the 81-bp rifampin resistance determining region (RRDR) and mutation V176F locating at the beginning of the ropB gene were analyzed by DNA sequencing of 86 Mycobacterium tuberculosis clinical isolates (72 resistant and 14 sensitive) from different parts of China. Sixty-five mutations of 22 distinct kinds, 21 point mutations, and 1 insertion were found in 65 of 72 resistant isolates. The most common mutations were in codons 531 (41%), 526 (40%), and 516 (4%). Mutations were not found in seven (10%) of the resistant isolates. Six new alleles within the RRDR, along with five novel mutations outside the RRDR, are reported. None of isolates contained the V176 mutation.

Alleles↗
PubMed2003

Using a cDNA microarray to study cellular gene expression altered by Mycobacterium tuberculosis.

OBJECTIVE: To examine the global effects of Mycobacterium tuberculosis (M. tuberculosis) infection on macrophages. METHODS: The gene expression profiling of macrophage U937, in response to infection with M. tuberculosis H(37)R(a), was monitored using a high-density cDNA microarray. RESULTS: M. tuberculosis infection caused 463 differentially expressed genes, of which 366 genes are known genes registered in the Gene Bank. These genes function in various cellular processes including intracellular signalling, cytoskeletal rearrangement, apoptosis, transcriptional regulation, cell surface receptors, cell-mediated immunity as well as a variety of cellular metabolic pathways, and may play key roles in M. tuberculosis infection and intracellular survival. CONCLUSIONS: M. tuberculosis infection alters the expression of host-cell genes, and these genes will provide a foundation for understanding the infection process of M. tuberculosis. The cDNA microarray is a powerful tool for studying pathogen-host cell interaction.

Cells, Cultured↗
PubMed2003

[Differential expression of human macrophage genes encoding cytokines and their regulatory elements after Mycobacterium tuberculosis infection].

To investigate whether the genes encoding cytokines and their regulatory elements participated in the immune response to Mycobacterium tuberculosis infection. Expression microarray was employed to compare the avirulent strain and clinically isolated strains infection induced macrophage cytokine differential expression. Results were cytokines IFN, TNF, TGF, IL and their regulatory elements are involved in the immune reaction. IL-19 was first reported to be involved in the anti-Mtb immunity. Relative expression level of theses factors before and after infection were assayed too. The data provides clues for further scrutinize the role of cytokines and related elements in the interaction between Mycobacterium tuberculosis and host macrophage.

Cytokines↗
PubMedDate not supplied

Titania and alumina sol-gel-derived microfluidics enzymatic-reactors for peptide mapping: design, characterization, and performance.

The design and characterization of titania-based and alumina-based Poly(dimethylsiloxane) (PDMS) microfluidics enzymatic-reactors along with their analytical features in coupling with MALDI-TOF and ESI-MS were reported. Microfluidics with microchannel and stainless steel tubing (SST) were fabricated using PDMS casting and O(2)-plasma techniques, and were used for the preparation of an enzymatic-reactor. Plasma oxidation for the PDMS microfluidic system enabled the channel wall of the microfluidics to present a layer of silanol (SiOH) groups. These SiOH groups act as anchors onto the microchannel wall linked covalently with the hydroxyl groups of trypsin-encapsulated sol matrix. As a result, the trypsin-encapsulated gel matrix was anchored onto the wall of the microchannel, and the leakage of gel matrix from the microchannel was effectively prevented. A feature of the microfluidic enzymatic-reactors is the feasibility of performing on-line protein analysis by attached SST electrode and replaceable tip. The success of trypsin encapsulation was investigated by AFM imaging, assay of enzymatic activity, CE detection, and MALDI-TOF and ESI-MS analysis. The lab-made devices provide an excellent extent of digestion even at a fast flow rate of 7.0 microL/min, which affords the very short residence time of ca. 2 s. With the present device, the digestion time was significantly shortened compared to conventional tryptic reaction schemes. In addition, the encapsulated trypsin exhibits increased stability even after continuous use. These features are required for high-throughput protein identification.

Aluminum Oxide↗