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Function of Lysine-148 in dTDP-D-Glucose 4,6-dehydratase from Streptomyces antibioticus Tu99
( Jae Kyung Sohng ),( Hyung Rae Noh ),( Oh Hyoung Lee ),( Sung Jun Kim ),( Ji Man Han ),( Seung Kwan Nam ),( Jin Cheol Yoo ) 한국미생물 · 생명공학회 2002 Journal of microbiology and biotechnology Vol.12 No.2
dTDP-D-glucose 4,6-dehydratase (TDPDH) catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, and requires NAD+ as a coenzyme for its catalytic activity. The dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus Tu99 tightly binds NAD+ [19]. In order to determine the role of lysine-148 in the NAD+ binding, the lysine of the dTDP-D-glucose 4,6-dehydratase from Streptomyces antibioticus Tu99 was mutated to various amino acids by site-directed mutagenesis. The catalytic activity of the four mutated enzymes of TDPDH did not recover after addition of NAD+. However, the activity of K159A, the mutated enzyme of UDP-D-glucose 4-epimerase (UDPE), recovered after the addition of NAD+ [15]. Although dTDP-glucose 4,6-dehydratase, and UDP-galactose (glucose) 4-epimerase are members of the short-chain dehydrogenase/reductase SDR family and the lysine-148 of TDPDH was highly conserved as in UDPE (Lys-l59), the function of the lysine-148 of TDPDH was different from that of UDPE. The mutated enzymes showed that the lysine-l48 of the dTDP-D-glucose 4,6-dehydratase played no role in the NAD+ binding. Accordingly, it is suggested that the lysine-l48 of the dTDP-D-glucose 4,6-dehydratase is involved in the folding of TDPDH.
Mechanism Study of dTDP-D-Glucose 4,6-Dehydratase: General Base in Active Site Domain
(Jae Kyung Sohng),(Hyung Rae Noh),(Jin Cheol Yoo) 생화학분자생물학회 1999 BMB Reports Vol.32 No.4
dTDP-D-glucose 4,6-dehydratase as an oxidoreductase catalyzes the conversion of dTDP-D-glucose to dTDP-4-keto-6-deoxy-D-glucose, which is essential for the formation of 6-deoxysugars. dTDP-D-glucose 4,6-dehydratase shows remarkable stereochemical convergence in which displacement of the C-6 hydroxyl group by a C-4 hydrogen proceeds intramolecularly with inversion of configuration. The reaction mechanism is known to be oxidation, dehydration, and reduction by bases mediating proton transfer and NAD^+ cofactor. In this study, the bases in the active site domain are proposed to be His-79 and His-300 from a comparison of the peptides of the dehydratase and UDP-D-glucose epimerase. His-79 and His-300 were mutated to prepare the mutants H79L (mutation of histidine to leucine at the 79th amino acid) and H300A (mutation of histidine to alanine at the 300th amino acid) by site-directed mutagenesis. The H79L protein was inactive, showing that His-79 participates in the reaction mechanism.
Jae Kyung Sohng,Jin Cheol Yoo 생화학분자생물학회 1996 BMB Reports Vol.29 No.3
DNA fragments, homologous to the dTDP-D-glucose 4,6-dehydratase gene, obtained from the genomic DNA of Streptomyces antibioticus Tu¨99, a producer of the unusual macrolide antibiotic chlorothricin, were cloned and sequenced. This dehydratase gene was designated as oxil. The coding region of the oxil gene is composed of 987 bp, and analysis of the DNA sequence data reveals sequences for the gene products of 329 amino acids (molecular weight of 36,037). The deduced amino acids are 59% identical to the StrE, dTDP-D-glucose 4,6-dehydratase from the streptomycin pathway. The oxil`s function was examined by expressing it in E. coli using the T7 RNA polymerase/promoter system (pRSET) to produce an active fusion protein including a his tag. This enzyme shows specificity of substrate, specific only to dTDPD-glucose.
Glycosylation of free sterol by whole-cell bioconversion in E. coli
Jae Kyung Sohng,Jae Kyung Sohng 한국당과학회 2011 한국당과학회 학술대회 Vol.2011 No.1
Steryl glucosides play important roles in many physiological and biochemical process in organism such as the heat shock, enhancement of immunological system, etc. The alignment of a putative sterol glucosides isolated from S. tropica CNB-440 has been shown 34%, 42% and 57% in homology with the corresponding ones from Arabidopis thaliana, Avena sativa and Salinispora arenicola CNS-205, respectively. Engineered E. coli host-high level production of the UDP-glucose was used for whole-cell bioconversion of free sterol (cholesterol and β-sistosterol) and the production of glycosylated product was only detected with β-sitosterol as substrate.