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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.

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Sequence alignments of ferredoxins and ferredoxin reductases.(A) Alignment of the amino acid sequence of Fd68 from S. ahygroscopicus ZB01 with different 3Fe-4S-Fds. Fd_rim(ZP_20965346) is from S. tubercidicus;Fd233(AY549200) is from S. tubercidicus R-922; Fd232(AY552101) is from S. tubercidicus I-1529; Fd_virT(WP_003995703) is from S. Viridochromogenes; Fd_virD(ZP_07308348) is from S. viridochromogenes DSM 40736; Fd_aur(ZP_10545293) is from S. auratus AGR0001; Fd_svi(ZP_06921672) is from S. sviceus ATCC 29083; Fd_cha(ZP_06921672) is from S. sviceus ATCC 29083; Fd_sp(BAG55293) is from Streptomyces sp. A-1544; Fd_amb(CAJ88533) is from S. ambofaciens ATCC 23877; Fd_gha(ZP_06574442) is from S. ghanaensis ATCC 14672. (B) Multiple sequence alignment of conserved regions in FdRs. Consensus sequences for the FAD ADP-binding motifs, the NAD (ADP)-binding motifs, and the FAD ribytil-binding motifs (Asturis 1995) are shown above the alignment. FdR18, FdR28 are the FdRs described in this study. FprD(NP_826852.1) is from S. avermitilis MA-4680. Fre3(AAT45306.1), Fre14(AAT45308.1), Fre16(AAT45309.1), FreEA(AAT45279.1) are from the genome of S. tubercidicus (Molnar, 2005). Fdr793(AJ628764.1) is of S. peucetius ATCC27952. SCF15(CAB60462.1) and SC4B10(CAC04223.1) are of S. coelicolor A3(2); FdR-sau(sau, EJJ04871.1) is of S. auratus AGR0001.
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pone-0098916-g002: Sequence alignments of ferredoxins and ferredoxin reductases.(A) Alignment of the amino acid sequence of Fd68 from S. ahygroscopicus ZB01 with different 3Fe-4S-Fds. Fd_rim(ZP_20965346) is from S. tubercidicus;Fd233(AY549200) is from S. tubercidicus R-922; Fd232(AY552101) is from S. tubercidicus I-1529; Fd_virT(WP_003995703) is from S. Viridochromogenes; Fd_virD(ZP_07308348) is from S. viridochromogenes DSM 40736; Fd_aur(ZP_10545293) is from S. auratus AGR0001; Fd_svi(ZP_06921672) is from S. sviceus ATCC 29083; Fd_cha(ZP_06921672) is from S. sviceus ATCC 29083; Fd_sp(BAG55293) is from Streptomyces sp. A-1544; Fd_amb(CAJ88533) is from S. ambofaciens ATCC 23877; Fd_gha(ZP_06574442) is from S. ghanaensis ATCC 14672. (B) Multiple sequence alignment of conserved regions in FdRs. Consensus sequences for the FAD ADP-binding motifs, the NAD (ADP)-binding motifs, and the FAD ribytil-binding motifs (Asturis 1995) are shown above the alignment. FdR18, FdR28 are the FdRs described in this study. FprD(NP_826852.1) is from S. avermitilis MA-4680. Fre3(AAT45306.1), Fre14(AAT45308.1), Fre16(AAT45309.1), FreEA(AAT45279.1) are from the genome of S. tubercidicus (Molnar, 2005). Fdr793(AJ628764.1) is of S. peucetius ATCC27952. SCF15(CAB60462.1) and SC4B10(CAC04223.1) are of S. coelicolor A3(2); FdR-sau(sau, EJJ04871.1) is of S. auratus AGR0001.

Mentions: A 1810 bp DNA fragment was cloned from S. ahygroscopicus ZB01 genome by PCR with primers F1/R1, and an open reading frame of 195 bp within the fragment was obtained and named fd68. The GC content of fd68 was as high as 71.3%. The deduced Fd68 contains 63 amino acids with a molecular weight of 7.1 kDa and was a putative [3Fe-4S] Fd, with the conserved amino acid binding sites coordinated with [3Fe-4S] type iron sulfur cluster [25] at Cys10, 16 and 54 (Fig. 2A). Fd68 exibits 89.1% identity and 88.9% similarity to Fd232 of S. tubercidicus I-529 and Fd233 of S. tubercidicus R-922 [5].


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)

Sequence alignments of ferredoxins and ferredoxin reductases.(A) Alignment of the amino acid sequence of Fd68 from S. ahygroscopicus ZB01 with different 3Fe-4S-Fds. Fd_rim(ZP_20965346) is from S. tubercidicus;Fd233(AY549200) is from S. tubercidicus R-922; Fd232(AY552101) is from S. tubercidicus I-1529; Fd_virT(WP_003995703) is from S. Viridochromogenes; Fd_virD(ZP_07308348) is from S. viridochromogenes DSM 40736; Fd_aur(ZP_10545293) is from S. auratus AGR0001; Fd_svi(ZP_06921672) is from S. sviceus ATCC 29083; Fd_cha(ZP_06921672) is from S. sviceus ATCC 29083; Fd_sp(BAG55293) is from Streptomyces sp. A-1544; Fd_amb(CAJ88533) is from S. ambofaciens ATCC 23877; Fd_gha(ZP_06574442) is from S. ghanaensis ATCC 14672. (B) Multiple sequence alignment of conserved regions in FdRs. Consensus sequences for the FAD ADP-binding motifs, the NAD (ADP)-binding motifs, and the FAD ribytil-binding motifs (Asturis 1995) are shown above the alignment. FdR18, FdR28 are the FdRs described in this study. FprD(NP_826852.1) is from S. avermitilis MA-4680. Fre3(AAT45306.1), Fre14(AAT45308.1), Fre16(AAT45309.1), FreEA(AAT45279.1) are from the genome of S. tubercidicus (Molnar, 2005). Fdr793(AJ628764.1) is of S. peucetius ATCC27952. SCF15(CAB60462.1) and SC4B10(CAC04223.1) are of S. coelicolor A3(2); FdR-sau(sau, EJJ04871.1) is of S. auratus AGR0001.
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pone-0098916-g002: Sequence alignments of ferredoxins and ferredoxin reductases.(A) Alignment of the amino acid sequence of Fd68 from S. ahygroscopicus ZB01 with different 3Fe-4S-Fds. Fd_rim(ZP_20965346) is from S. tubercidicus;Fd233(AY549200) is from S. tubercidicus R-922; Fd232(AY552101) is from S. tubercidicus I-1529; Fd_virT(WP_003995703) is from S. Viridochromogenes; Fd_virD(ZP_07308348) is from S. viridochromogenes DSM 40736; Fd_aur(ZP_10545293) is from S. auratus AGR0001; Fd_svi(ZP_06921672) is from S. sviceus ATCC 29083; Fd_cha(ZP_06921672) is from S. sviceus ATCC 29083; Fd_sp(BAG55293) is from Streptomyces sp. A-1544; Fd_amb(CAJ88533) is from S. ambofaciens ATCC 23877; Fd_gha(ZP_06574442) is from S. ghanaensis ATCC 14672. (B) Multiple sequence alignment of conserved regions in FdRs. Consensus sequences for the FAD ADP-binding motifs, the NAD (ADP)-binding motifs, and the FAD ribytil-binding motifs (Asturis 1995) are shown above the alignment. FdR18, FdR28 are the FdRs described in this study. FprD(NP_826852.1) is from S. avermitilis MA-4680. Fre3(AAT45306.1), Fre14(AAT45308.1), Fre16(AAT45309.1), FreEA(AAT45279.1) are from the genome of S. tubercidicus (Molnar, 2005). Fdr793(AJ628764.1) is of S. peucetius ATCC27952. SCF15(CAB60462.1) and SC4B10(CAC04223.1) are of S. coelicolor A3(2); FdR-sau(sau, EJJ04871.1) is of S. auratus AGR0001.
Mentions: A 1810 bp DNA fragment was cloned from S. ahygroscopicus ZB01 genome by PCR with primers F1/R1, and an open reading frame of 195 bp within the fragment was obtained and named fd68. The GC content of fd68 was as high as 71.3%. The deduced Fd68 contains 63 amino acids with a molecular weight of 7.1 kDa and was a putative [3Fe-4S] Fd, with the conserved amino acid binding sites coordinated with [3Fe-4S] type iron sulfur cluster [25] at Cys10, 16 and 54 (Fig. 2A). Fd68 exibits 89.1% identity and 88.9% similarity to Fd232 of S. tubercidicus I-529 and Fd233 of S. tubercidicus R-922 [5].

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.

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