Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bact... more Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r(2) of 0.90-0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by -1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C-H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P-O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure-function relationship of biological DNA-phosphorothiation.
... 1 Tetrahedron Letters 2,3,6-Trideoxy Sugar Nucleotides: Synthesis and Stability Mingxuan Wua,... more ... 1 Tetrahedron Letters 2,3,6-Trideoxy Sugar Nucleotides: Synthesis and Stability Mingxuan Wua,c, Qingqing Menga, Min Geb, Linquan Baic and ... Phosphates 12b and 12c were successfully converted to their UDP and TDP derivatives using the method developed by Wong et al ...
FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Strep... more FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Streptomyces sp. FR-008 as a complex mixture of compounds. Here, six components (FR-008-I to -VI) of the FR-008/candicidin complex were determined; III, V, and VI were confirmed as natural products, principally differing from each other at C-3 and C-9, while the other three were believed to originate from the respective conversions of the natural ones in vitro. Inactivation of KR21 and DH18, respectively, abolished production of V carrying a C-3 hydroxyl, and VI carrying a C-9 methylene. Combined inactivation created a mutant producing only III, with a C-3 ketone and a C-9 hydroxyl, and having antifungal activity superior to V and comparable to VI. Incomplete activities of KR21 and DH18 were, therefore, unambiguously identified as being involved in structural variations of FR-008 complex.
In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also ... more In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also known as candicidin, consists of twenty-one genes, including four regulatory genes, fscRI to fscRIV. Our bioinformatics analyses indicate that FscRI has an N-terminal PAS domain, whereas the other three regulators have N-terminal AAA domains and are members of the LAL (large ATP-binding regulators of the LuxR type) family. Deletion of fscRI abolished the production of FR-008, with production restored in the complemented strain, supporting a critical role for FscRI in FR-008 biosynthesis. Consistent with these findings, transcription of genes involved in the biosynthesis and efflux of FR-008 was greatly downregulated in ΔfscRI. Interestingly, the regulatory gene fscRIV was also downregulated in ΔfscRI. Production of FR-008 was reduced, but not abrogated, in an fscRIV deletion mutant, and although structural genes were downregulated in ΔfscRIV, the changes were much less dramatic than in Δ...
Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bact... more Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r(2) of 0.90-0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by -1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C-H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P-O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure-function relationship of biological DNA-phosphorothiation.
A new compound of ansamitocin was isolated from the extracts of fermentation medium of mutant str... more A new compound of ansamitocin was isolated from the extracts of fermentation medium of mutant strain HGF052 derived from Actinosynnema pretiosum ssp. aurantium ATCC 31565, and identified as N-demethyl-desepoxy-9-methoxy-maytansinol (1) on the basis of extensive spectroscopic methods. Bioassay results showed that compound 1 had cytotoxic activity against HL-60 and BEL-7402 cell lines.
This review describes the recent research activities in China in relation to studies on antibioti... more This review describes the recent research activities in China in relation to studies on antibiotic biosynthetic pathways and pathway engineering in actinomycetes. 75 references are cited.
Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated deriva... more Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the clt cluster encoding cyclothiazomycin from Streptomyces hygroscopicus 10-22 was revealed by genome sequencing in S. hygroscopicus 5008. Genes SHJG8833 and SHJG8837 of the cluster and flanking gene SHJG8838 were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that SHJG8833, but not SHJG8837 or SHJG8838, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of SHJG8833 was located to a thymidine 54 nt upstream of the start codon. Inactivation of SHJG8833 abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing SHJG8833 into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from SHJG8826 to SHJG8832, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons, SHJG8826-27 and SHJG8828-32. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of SHJG8826 and SHJG8828 and sequences internal to SHJG8826 and SHJG8829, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the clt cluster.
Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bact... more Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r(2) of 0.90-0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by -1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C-H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P-O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure-function relationship of biological DNA-phosphorothiation.
... 1 Tetrahedron Letters 2,3,6-Trideoxy Sugar Nucleotides: Synthesis and Stability Mingxuan Wua,... more ... 1 Tetrahedron Letters 2,3,6-Trideoxy Sugar Nucleotides: Synthesis and Stability Mingxuan Wua,c, Qingqing Menga, Min Geb, Linquan Baic and ... Phosphates 12b and 12c were successfully converted to their UDP and TDP derivatives using the method developed by Wong et al ...
FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Strep... more FR-008/candicidin is a heptaene macrolide with established antifungal activity, produced by Streptomyces sp. FR-008 as a complex mixture of compounds. Here, six components (FR-008-I to -VI) of the FR-008/candicidin complex were determined; III, V, and VI were confirmed as natural products, principally differing from each other at C-3 and C-9, while the other three were believed to originate from the respective conversions of the natural ones in vitro. Inactivation of KR21 and DH18, respectively, abolished production of V carrying a C-3 hydroxyl, and VI carrying a C-9 methylene. Combined inactivation created a mutant producing only III, with a C-3 ketone and a C-9 hydroxyl, and having antifungal activity superior to V and comparable to VI. Incomplete activities of KR21 and DH18 were, therefore, unambiguously identified as being involved in structural variations of FR-008 complex.
In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also ... more In Streptomyces sp. FR-008, the biosynthetic gene cluster of the polyene antibiotic FR-008, also known as candicidin, consists of twenty-one genes, including four regulatory genes, fscRI to fscRIV. Our bioinformatics analyses indicate that FscRI has an N-terminal PAS domain, whereas the other three regulators have N-terminal AAA domains and are members of the LAL (large ATP-binding regulators of the LuxR type) family. Deletion of fscRI abolished the production of FR-008, with production restored in the complemented strain, supporting a critical role for FscRI in FR-008 biosynthesis. Consistent with these findings, transcription of genes involved in the biosynthesis and efflux of FR-008 was greatly downregulated in ΔfscRI. Interestingly, the regulatory gene fscRIV was also downregulated in ΔfscRI. Production of FR-008 was reduced, but not abrogated, in an fscRIV deletion mutant, and although structural genes were downregulated in ΔfscRIV, the changes were much less dramatic than in Δ...
Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bact... more Phosphorothioation, with sulfur replacing a nonbridging oxygen of phosphate, has surfaced in bacterial DNA electrophoresis. To understand structural characteristics of the thio-substituted DNA, we have investigated the correlation between the relative energy of phosphate/phosphorothioate linkage and the backbone torsions. The relative energies (R.E.) computed by the quantum mechanical method, the PBE1PBE(CPCM, solvent=water)//PBE1PBE/6-31+G(2df) level of theory, were used to construct energy-scoring functions against backbone torsion variables, resulting in the squared correlation coefficients r(2) of 0.90-0.95. Then, the DNA energy alteration by phosphorothioation is estimated with the relative energy difference (ΔR.E.) between phosphate and phosphorothioate of the phosphate linkages in the DNA crystallographic database (NDB). As a result, Rp-phosphorothioation shifts the relative energy of B-helical structures by 2.7 ± 3.4 kcal/mol, destabilizing about 95% linkages, while Sp-phosphorothioation by -1.4 ± 2.4 kcal/mol, stabilizing over 84% linkages in the data sets. The B-helical destabilization is likely caused by the steric effect between the sulfur atom of Rp-phosphorothioate and the neighboring C-H groups of deoxyribose on the groove wall in B-helix. The unfavorable interaction may be magnified by the increasing rigidness of P-O-involving backbone torsions α and ζ upon the nonbridging phosphorothioations. Since B-helix is the most prevalent DNA double-helical structure and Rp-phosphorothioation is the exclusive configuration in bacteria thio-DNA found to date, the observed stereospecificity-destabilization correlation may reflect a structure-function relationship of biological DNA-phosphorothiation.
A new compound of ansamitocin was isolated from the extracts of fermentation medium of mutant str... more A new compound of ansamitocin was isolated from the extracts of fermentation medium of mutant strain HGF052 derived from Actinosynnema pretiosum ssp. aurantium ATCC 31565, and identified as N-demethyl-desepoxy-9-methoxy-maytansinol (1) on the basis of extensive spectroscopic methods. Bioassay results showed that compound 1 had cytotoxic activity against HL-60 and BEL-7402 cell lines.
This review describes the recent research activities in China in relation to studies on antibioti... more This review describes the recent research activities in China in relation to studies on antibiotic biosynthetic pathways and pathway engineering in actinomycetes. 75 references are cited.
Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated deriva... more Cyclothiazomycin is a member of the thiopeptide antibiotics, which are usually complicated derivatives of ribosomally synthesized peptides. A gene cluster containing 12 ORFs identical to the clt cluster encoding cyclothiazomycin from Streptomyces hygroscopicus 10-22 was revealed by genome sequencing in S. hygroscopicus 5008. Genes SHJG8833 and SHJG8837 of the cluster and flanking gene SHJG8838 were predicted to encode regulatory proteins from different families. In this study, we showed that the newly identified cluster is functional and we investigated the roles of these regulatory genes in the regulation of cyclothiazomycin biosynthesis. We determined that SHJG8833, but not SHJG8837 or SHJG8838, is critical for cyclothiazomycin biosynthesis. The transcriptional start point of SHJG8833 was located to a thymidine 54 nt upstream of the start codon. Inactivation of SHJG8833 abrogated the production of cyclothiazomycin, and synthesis could be restored by reintroducing SHJG8833 into the mutant strain. Gene expression analyses indicated that SHJG8833 regulates a consecutive set of seven genes from SHJG8826 to SHJG8832, whose products are predicted to be involved in different steps in the construction of the main framework of cyclothiazomycin. Transcriptional analysis indicated that these seven genes may form two operons, SHJG8826-27 and SHJG8828-32. Gel-shift analysis demonstrated that the DNA-binding domain of SHJG8833 binds the promoters of SHJG8826 and SHJG8828 and sequences internal to SHJG8826 and SHJG8829, and a conserved binding sequence was deduced. These results indicate that SHJG8833 is a positive regulator that controls cyclothiazomycin biosynthesis by activating structural genes in the clt cluster.
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