Sukhoon Koh

Application of rpoB and Zinc Protease Gene for Use in Molecular Discrimination of Fusobacterium nucleatum Subspecies

ABSTRACT Fusobacterium nucleatum is classified into five subspecies that inhabit the human oral cavity (F. nucleatum subsp. nucleatum, F. nucleatum subsp. polymorphum, F. nucleatum subsp. fusiforme, F. nucleatum subsp. vincentii, and F. nucleatum subsp. animalis) based on several phenotypic characteristics and DNA-DNA hybridization patterns. However, the methods for detecting or discriminating the clinical isolates of F. nucleatum at the subspecies levels are laborious, expensive, and time-consuming. Therefore, in this study, the nucleotide sequences of the RNA polymerase β-subunit gene (rpoB) and zinc protease gene were analyzed to discriminate the subspecies of F. nucleatum. The partial sequences of rpoB (approximately 2,419 bp), the zinc protease gene (878 bp), and 16S rRNA genes (approximately 1,500 bp) of the type strains of five subspecies, 28 clinical isolates of F. nucleatum, and 10 strains of F. periodonticum (as a control group) were determined and analyzed. The phylogenetic data showed that the rpoB and zinc protease gene sequences clearly delineated the subspecies of F. nucleatum and provided higher resolution than the 16S rRNA gene sequences in this respect. According to the phylogenetic analysis of rpoB and the zinc protease gene, F. nucleatum subsp. vincentii and F. nucleatum subsp. fusiforme might be classified into a single subspecies. Five clinical isolates could be delineated as a new subspecies of F. nucleatum. The results suggest that rpoB and the zinc protease gene are efficient targets for the discrimination and taxonomic analysis of the subspecies of F. nucleatum.

Trehalose synthesis from maltose by a thermostable trehalose synthase from Thermus caldophilus

Purified trehalose synthase from Thermus caldophilus GK24 produced 18–86% trehalose from 10 mM–1 M maltose. The enzyme also catalyzed the conversion of α,α-trehalose into maltose but did not act on other disaccharides. The yield of trehalose from maltose by this enzyme increased 30% more at 40°C than at 80°C and was independent of the substrate concentration. The maximum yield of α,α-trehalose from 10 mM maltose reached 86% at 40°C. In addition, α,β-trehalose was also formed from maltose or α,α-trehalose at 3.5% yield at 80°C.

A practical enzymatic synthesis of UDP sugars and NDP glucoses.

Nucleoside diphosphate (NDP) sugar pyrophosphorylases (NPs) catalyze the bioconversion of sugar-1-phosphates (Su1Ps) and nucleoside triphosphates (NTPs) to NDP sugars. Various glycosyltransferases specifically transfer these activated monosaccharide residues onto a free hydroxyl or amino group on an acceptor molecule to synthesize bioactive metabolites such as glycogen, trehalose, lipopolysaccharides, and peptidoglycans, which are present in the bacterial cell wall. Furthermore, many pharmaceutically relevant natural products are glycosylated secondary metabolites, which are thought to have potential for the development of novel glycorandomization therapeutics. Although chemical syntheses of a few NDP sugars have been reported by various groups, the methods used were complicated by the laborious maneuvering of protecting groups in order to achieve regioselectivity. By contrast, NPs from natural sources catalyze regioselective phosphorylation by modulation of the metabolic intermediates. For these reasons, a variety of in vitro synthetic routes to NDP sugars by a salvage or a de novo pathway have been designed and implemented. In view of the synthetic availability of high-energy donor NDP sugars for glycosyltransferases, we report the synthesis of UDP sugars and NDP glucoses by recombinant Thermus caldophilus GK24 (Tca) UDP-sugar pyrophosphorylase (UP) and the substrate specificity of the enzyme toward various NTPs and Su1Ps. The Tca usp gene (GeneBank accession no. : AAV80705), which encodes UP, was cloned and inserted into the pKK223-3 vector to construct plasmid pGLM. The tetrameric UP protein was overexpressed at an optimal temperature of 70 8C in Escherichia coli MV1184 harboring pGLM (see the Supporting Information), and purified by heat precipitation and one-step anion-exchange column chromatography (Figure 1). The presence of Mg and Mn at an appropriate concentration was required for Tca UP activity; Co 2+ and Ni were less effective (see the Supporting Information). A thermostability test showed that Tca UP was very stable at 70 8C for 1 h (see the Supporting Information).

Biochemical characterization of a UDP‐sugar pyrophosphorylase from Thermus caldophilus GK24

An extremely thermostable UDP‐GlcNAc pyrophosphorylase has been purified from Thermus caldophilus GK24 by chromatographic methods including ion‐exchange, hydrophobic interaction, and affinity chromatographies. The specific activity of the enzyme was enriched 41.8‐fold, with a recovery of 2%. The molecular mass of the enzyme was 41 kDa by SDS/PAGE and 45 kDa by gel‐filtration chromatography. The activity was maximum at 86 °C and its half‐life at 95 °C was 30 min. Its optimum pH was 6.9 in the presence of Mg2+ ions. A biochemical study showed that UDP‐GlcNAc pyrophosphorylase activity could be enhanced by fructose 1‐phosphate, a precursor of UDP‐GlcNAc. The enzyme showed a broad substrate specificity with sugar 1‐phosphates, including glucose 1‐phosphate, GlcNAc‐1‐P and xylose 1‐phosphate. The enzyme was therefore named UDP‐sugar pyrophosphorylase. The N‐terminal and internal peptide sequences were determined and compared with known sequences from various sources. It was found that N‐terminal sequence is similar to that of UDP‐GlcNAc and UDP‐glucose pyrophosphorylases from other bacterial sources.

Cloning and expression of the gene for xylose isomerase from Thermus flavus AT62 in Escherichia coli

The gene encoding xylose isomerase (xylA) was cloned fromThermus flavus AT62 and the DNA sequence was determined. ThexylA gene encodes the enzyme xylose isomerase (XI orxylA) consisting of 387 amino acids (calculated Mr of 44,941). Also, there was a partial xylulose kinase gene that was 4 bp overlapped in the end of XI gene. The XI gene was stably expressed inE. coli under the control oftac promoter. XI produced inE. coli was simply purified by heat treatment at 90°C for 10 min and column chromatography of DEAE-Sephacel. The Mr of the purified enzyme was estimated to be 45 kDa on SDS-polyacrylamide gel electrophoresis. However, Mr of the cloned XI was 185 kDa on native condition, indicating that the XI consists of homomeric tetramer. The enzyme has an optimum temperature at 90°C. Thermostability tests revealed that half life at 85°C was 2 mo and 2 h at 95°C. The optimum pH is around 7.0, close to where by-product formation is minimal. The isomerization yield of the cloned XI was about 55% from glucose, indicating that the yield is higher than those of reported enzymes. The Km values for various sugar substrates were calculated as 106 mM for glucose. Divalent cations such as Mn2+, Co2+, and Mg2+ are required for the enzyme activity and 100 mM EDTA completely inhibited the enzyme activity.

Identification of novel allele on the locus 47z ( DXYS5 ) in the Korean population

AbstractTwo homogenous sequences of 47z (DXYS5) are located on the X (DXYS5X) and Y (DXYS5Y) chromosomes, and these are known to be useful polymorphic markers for tracing male-specific gene flow such as the migration routes of human populations. Using the 47z/StuI PCR-RFLP system, we found a novel allele which showed two bands, in contrast to the previous two allele types, one band (Y1) and three bands (Y2). This means that copies of PCR products derived from both the DXYS5X and DXYS5Y loci were clearly cut by the StuI enzyme, implying that the DXYS5X locus of the X chromosome is polymorphic. Allelic frequencies examined in 267 male Korean individuals showed that 95.8% had Y1, 3.4% Y2, and 0.8% had the novel allele. Our findings should contribute to a better understanding of genetic polymorphism on X and Y chromosomes, the molecular evolution mechanism of sex chromosomes, and how the migration route of Koreans is related to those of other East Asian populations.