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Diversity of Cytochrome P450 in Streptomyces peucetius
Parajuli, Niranjan,Basnet, Devi B.,Kim, Byung-Gee,Lee, Hei Chan,Sohng, Jae Kyung,Liou, Kwangkyoung 한국공업화학회 2004 응용화학 Vol.8 No.1
We have determined the genome sequences of 8.7 Mb chromosome of Streptomyces peucetiusATCC 27952, which produces clinically important anthracyline chemotherapeutic agents of the polyketide class of antibiotics, daunorubicin and doxorubicin. The cytochrome P450 gene superfamily is represented by 19 sequences in the S. peucetius. Amongthose, 15 code for apparently functional genes whereas four are apparently pseudo genes. Four cytochrome P450s are associated with modular PKS of avermectin, and two with doxorubicin biosynthetic gene cluster. CYP252A1 is the new family found in S. peucetius, which shares 38% identity to CYP51 from Streptomyces coelicolor A3 (2).
Parajuli, Niranjan,Basnet, Devi B.,Chung, Young Soo,Lee, Hei Chan,Liou, Kwangkyoung,Sohng, Jae Kyung Elsevier 2005 Enzyme and microbial technology Vol.37 No.4
<P><B>Abstract</B></P><P>We have found that the thermophilic activity of glucose-1-phosphate thymidylyltransferase (stRmlA) is conditional upon the presence certain substances. In particular, it showed its thermal stability only in the presence of 50mM dTTP or dTMP, thermal stability being the highest at 70°C. The purified enzyme was stable up to 90°C within a broad pH range from 2.0 to 13.0, and its maximum activity was measured at a pH of 11.5 at 70°C. Unlike other mesophilic counterparts, it showed catalytic activity in the presence of various metal ions in the following order of reactivity: Mg<SUP>2+</SUP>>Zn<SUP>2+</SUP>>Cu<SUP>2+</SUP>>Co<SUP>2+</SUP>>Fe<SUP>2+</SUP>>Ca<SUP>2+</SUP>>Fe<SUP>3+</SUP>>Ni<SUP>2+</SUP>. Its catalytic activity was not inhibited even by the denaturants 50mM guanidine hydrochloride and 50mM urea.</P><P>To explore the molecular basis for its unusual thermostability, homology structural modeling, codon usage comparisons, and amino acid composition analyses were performed. The CCC, CUC and UCC codons are significantly higher, and distributed uniformly in <I>strmlA</I> as contrasted with other GPTTs. Our analyses revealed that the GC rich coding sequences may not be the reason for thermostability of stRmlA. The β-sheets are also found more likely in stRmlA, which contributes to thermal stability by increasing the number of hydrogen bonds. Further results suggest that a large number of apolar functional groups exposed to solvent accessible surface area, a significant number of residues sensitive to oxidation or deamination, and a higher hydrophobicity, any or all of the which could be reasons for its unique thermophilic behavior. Finally, we postulate that dTTP or dTMP enters the active site and binds tightly to inhibit the defolding of the protein, which, in turn, increases its thermal stability. Such findings will be useful for further investigations on thermophilic behavior of enzymes.</P>
Probing 3-Hydroxyflavone for <i>In Vitro</i> Glycorandomization of Flavonols by YjiC
Pandey, Ramesh Prasad,Parajuli, Prakash,Koirala, Niranjan,Park, Je Won,Sohng, Jae Kyung American Society for Microbiology 2013 Applied and environmental microbiology Vol.79 No.21
<P>The glycosylation of five different flavonols, fisetin, quercetin, myricetin, kaempferol, and 3-hydroxyflavone, was achieved by applying YjiC. 3-Hydroxyflavone was selected as a probe for <I>in vitro</I> glycorandomization of all flavonols using diverse nucleotide diphosphate-<SMALL>d/l</SMALL>-sugars. This study unlocked the possibilities of the glycodiversification of flavonols and the generation of novel compounds as future therapeutics.</P>
Glucosylation of Isoflavonoids in Engineered Escherichia coli
Pandey, Ramesh Prasad,Parajuli, Prakash,Koirala, Niranjan,Lee, Joo Ho,Park, Yong Il,Sohng, Jae Kyung Korean Society for Molecular and Cellular Biology 2014 Molecules and cells Vol.37 No.2
A glycosyltransferase, YjiC, from Bacillus licheniformis has been used for the modification of the commercially available isoflavonoids genistein, daidzein, biochanin A and formononetin. The in vitro glycosylation reaction, using UDP-${\alpha}$-D-glucose as a donor for the glucose moiety and aforementioned four acceptor molecules, showed the prominent glycosylation at 4' and 7 hydroxyl groups, but not at the $5^{th}$ hydroxyl group of the A-ring, resulting in the production of genistein 4'-O-${\beta}$-D-glucoside, genistein 7-O-${\beta}$-D-glucoside (genistin), genistein 4',7-O-${\beta}$-D-diglucoside, biochanin A-7-O-${\beta}$-D-glucoside (sissotrin), daidzein 4'-O-${\beta}$-D-glucoside, daidzein 7-O-${\beta}$-D-glucoside (daidzin), daidzein 4', 7-O-${\beta}$-D-diglucoside, and formononetin 7-O-${\beta}$-D-glucoside (ononin). The structures of all the products were elucidated using high performance liquid chromatography-photo diode array and high resolution quadrupole time-of-flight electrospray ionization mass spectrometry (HR QTOF-ESI/MS) analysis, and were compared with commercially available standard compounds. Significantly higher bioconversion rates of all four isoflavonoids was observed in both in vitro as well as in vivo bioconversion reactions. The in vivo fermentation of the isoflavonoids by applying engineered E. coli $BL21(DE3)/{\Delta}pgi{\Delta}zwf{\Delta}ushA$ overexpressing phosphoglucomutase (pgm) and glucose 1-phosphate uridyltransferase (galU), along with YjiC, found more than 60% average conversion of $200{\mu}M$ of supplemented isoflavonoids, without any additional UDP-${\alpha}$-D-glucose added in fermentation medium, which could be very beneficial to large scale industrial production of isoflavonoid glucosides.