Limits...
Exploring the electron transfer pathway in the oxidation of avermectin by CYP107Z13 in Streptomyces ahygroscopicus ZB01.

Li M, Zhang Y, Zhang L, Yang X, Jiang X - PLoS ONE (2014)

Bottom Line: Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin.A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin.Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin. CYP107Z13 is responsible for this site-specific oxidation in ZB01. In the present study, we explored the electron transfer pathway in oxidation of avermectin by CYP107Z13 in ZB01. A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin. To clarify whether FdR18 and FdR28 participate in the electron transfer during avermectin oxidation by CYP107Z13, two whole-cell biocatalytic systems were designed in E. coli BL21 (DE3), with one co-expressing CYP107Z13, Fd68 and FdR18 and the other co-expressing CYP107Z13, Fd68 and FdR28. Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin. Thus, we propose an electron transfer pathway NADH→FdR18/FdR28→Fd68→CYP107Z13 for oxidation of avermectin to form 4″-oxo-avermectin in ZB01.

Show MeSH
Expression and characterization of FdR18 and FdR28.(A) Recombinant expression vectors pRSET-fdr18 and pRSET-fdr28. (B) SDS-PAGE analysis of recombinant proteins FdR18 and FdR28 expressed by E. coli BL21 (DE3). Mr: protein markers. (C) UV-visible spectra of purified FdR18 and FdR28. Spectra were recorded at ambient temperature in 50 mM Tris buffer (pH 7.5). (D) DCPIP reduction activities of purified FdR18 and FdR28, measured in the presence of 200 uM NADH (▪) or NADPH (□).
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4048220&req=5

pone-0098916-g004: Expression and characterization of FdR18 and FdR28.(A) Recombinant expression vectors pRSET-fdr18 and pRSET-fdr28. (B) SDS-PAGE analysis of recombinant proteins FdR18 and FdR28 expressed by E. coli BL21 (DE3). Mr: protein markers. (C) UV-visible spectra of purified FdR18 and FdR28. Spectra were recorded at ambient temperature in 50 mM Tris buffer (pH 7.5). (D) DCPIP reduction activities of purified FdR18 and FdR28, measured in the presence of 200 uM NADH (▪) or NADPH (□).

Mentions: pRSET-fdr18 and pRSET-fdr28 (Fig. 4A) were constructed and transformed into E. coli BL21(DE3), the resultant transformants were named E. coli-fdr18 and E. coli-fdr28 respectively. The recombinant FdR18 and FdR28 proteins were then expressed and purified. The molecular weight of FdR28 was greater than FdR18 on SDS-PAGE (Fig. 4B). UV-visible spectra analysis demonstrated that absorption peaks appeared at 388, 453, and 482 nm for oxidized FdR18 and at 386, 455, and 486 nm for FdR28 (Fig. 4C). The electron transport rates of FdR18 and FdR28 for NADH and NADPH were detected using DCPIP as the electron acceptor. The Km and Kcat of FdR18 for NADH, evaluated using DCPIP, was 64 µM and 121 min−1, whereas those of FdR28 for NADH were 25.4 µM and 386 min–1, respectively. Both FdR18 and FdR28 proteins showed higher electron transport activity against NADH than NADPH, showing that both of the proteins are possible NADH-dependent FdRs (Fig. 4D).


Exploring the electron transfer pathway in the oxidation of avermectin by CYP107Z13 in Streptomyces ahygroscopicus ZB01.

Li M, Zhang Y, Zhang L, Yang X, Jiang X - PLoS ONE (2014)

Expression and characterization of FdR18 and FdR28.(A) Recombinant expression vectors pRSET-fdr18 and pRSET-fdr28. (B) SDS-PAGE analysis of recombinant proteins FdR18 and FdR28 expressed by E. coli BL21 (DE3). Mr: protein markers. (C) UV-visible spectra of purified FdR18 and FdR28. Spectra were recorded at ambient temperature in 50 mM Tris buffer (pH 7.5). (D) DCPIP reduction activities of purified FdR18 and FdR28, measured in the presence of 200 uM NADH (▪) or NADPH (□).
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4048220&req=5

pone-0098916-g004: Expression and characterization of FdR18 and FdR28.(A) Recombinant expression vectors pRSET-fdr18 and pRSET-fdr28. (B) SDS-PAGE analysis of recombinant proteins FdR18 and FdR28 expressed by E. coli BL21 (DE3). Mr: protein markers. (C) UV-visible spectra of purified FdR18 and FdR28. Spectra were recorded at ambient temperature in 50 mM Tris buffer (pH 7.5). (D) DCPIP reduction activities of purified FdR18 and FdR28, measured in the presence of 200 uM NADH (▪) or NADPH (□).
Mentions: pRSET-fdr18 and pRSET-fdr28 (Fig. 4A) were constructed and transformed into E. coli BL21(DE3), the resultant transformants were named E. coli-fdr18 and E. coli-fdr28 respectively. The recombinant FdR18 and FdR28 proteins were then expressed and purified. The molecular weight of FdR28 was greater than FdR18 on SDS-PAGE (Fig. 4B). UV-visible spectra analysis demonstrated that absorption peaks appeared at 388, 453, and 482 nm for oxidized FdR18 and at 386, 455, and 486 nm for FdR28 (Fig. 4C). The electron transport rates of FdR18 and FdR28 for NADH and NADPH were detected using DCPIP as the electron acceptor. The Km and Kcat of FdR18 for NADH, evaluated using DCPIP, was 64 µM and 121 min−1, whereas those of FdR28 for NADH were 25.4 µM and 386 min–1, respectively. Both FdR18 and FdR28 proteins showed higher electron transport activity against NADH than NADPH, showing that both of the proteins are possible NADH-dependent FdRs (Fig. 4D).

Bottom Line: Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin.A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin.Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing, China.

ABSTRACT
Streptomyces ahygroscopicus ZB01 can effectively oxidize 4″-OH of avermectin to form 4″-oxo-avermectin. CYP107Z13 is responsible for this site-specific oxidation in ZB01. In the present study, we explored the electron transfer pathway in oxidation of avermectin by CYP107Z13 in ZB01. A putative [3Fe-4S] ferredoxin gene fd68 and two possible NADH-dependent ferredoxin reductase genes fdr18 and fdr28 were cloned from the genomic DNA of ZB01. fd68 gene disruption mutants showed no catalytic activity in oxidation of avermectin to form 4″-oxo-avermectin. To clarify whether FdR18 and FdR28 participate in the electron transfer during avermectin oxidation by CYP107Z13, two whole-cell biocatalytic systems were designed in E. coli BL21 (DE3), with one co-expressing CYP107Z13, Fd68 and FdR18 and the other co-expressing CYP107Z13, Fd68 and FdR28. Both of the two biocatalytic systems were found to be able to mediate the oxidation of avermectin to form 4″-oxo-avermectin. Thus, we propose an electron transfer pathway NADH→FdR18/FdR28→Fd68→CYP107Z13 for oxidation of avermectin to form 4″-oxo-avermectin in ZB01.

Show MeSH