A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii

Blanco, Gloria; Pereda, Ana; Brian, Paul; Méndez, Cármen; Chater, Keith F.; Salas, José A.

doi:10.1128/jb.175.24.8043-8048.1993

Cited by 23 publications

(21 citation statements)

References 26 publications

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“…strain EST1001; TodA [50], ferredoxin oxidoreductase of toluene dioxygenase from P. putida F1; BphG [10], ferredoxin oxidoreductase of biphenyl dioxygenase from Pseudomonas strain LB400; PcpB [28], pentachlorophenol-4-monooxygenase from Flavobacterium sp. strain ATCC 39723; TcmG [6], tetracenomycin A2 hydroxylase from Streptomyces glaucescens; RdmE [24], putative aklavinone-11-hydroxylase from Streptomyces purpurascens; SchC [4], putative hydroxylase involved in spore pigment synthesis from S. halstedii; DnrF [12], putative aklavinone-11-hydroxylase from Streptomyces paucetius; Chl2 [5], tetracycline 6-hydroxylase from Streptomyces aureofaciens). In the fingerprint of the ADP binding motif (A) proposed by Wierenga et al (46), ⍀ represents the amino acids H, K, N, Q, R, S, and T; ⌬ represents A, C, I, L, M, and V; and is D and E. The short sequence important in FAD binding for many FAD-containing enzymes (8,30) is shown in panel B.…”

Section: Discussionmentioning

confidence: 99%

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Haigler¹,

Suen²,

Spain³

1996

J Bacteriol

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4-Methyl-5-nitrocatechol (MNC) is an intermediate in the degradation of 2,4-dinitrotoluene byBurkholderia sp. strain DNT. In the presence of NADPH and oxygen, MNC monooxygenase catalyzes the removal of the nitro group from MNC to form 2-hydroxy-5-methylquinone. The gene (dntB) encoding MNC monooxygenase has been previously cloned and characterized. In order to examine the properties of MNC monooxygenase and to compare it with other enzymes, we sequenced the gene encoding the MNC monooxygenase and purified the enzyme from strain DNT. dntB was localized within a 2.2-kb ApaI DNA fragment. Sequence analysis of this fragment revealed an open reading frame of 1,644 bp with an N-terminal amino acid sequence identical to that of purified MNC monooxygenase from strain DNT. Comparison of the derived amino acid sequences with those of other genes showed that DntB contains the highly conserved ADP and flavin adenine dinucleotide (FAD) binding motifs characteristic of flavoprotein hydroxylases. MNC monooxygenase was purified to homogeneity from strain DNT by anion exchange and gel filtration chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein with a molecular weight of 60,200, which is consistent with the size determined from the gene sequence. The native molecular weight determined by gel filtration was 65,000, which indicates that the native enzyme is a monomer. It used either NADH or NADPH as electron donors, and NADPH was the preferred cofactor. The purified enzyme contained 1 mol of FAD per mol of protein, which is also consistent with the detection of an FAD binding motif in the amino acid sequence of DntB. MNC monooxygenase has a narrow substrate specificity. MNC and 4-nitrocatechol are good substrates whereas 3-methyl-4-nitrophenol, 3-methyl-4-nitrocatechol, 4-nitrophenol, 3-nitrophenol, and 4-chlorocatechol were not. These studies suggest that MNC monooxygenase is a flavoprotein that shares some properties with previously studied nitrophenol oxygenases.Microorganisms can use either monooxygenase (33,34,36,49) or dioxygenase (2,15,21,25,33,37,40) enzymes to catalyze the oxidative removal of nitro groups from nitroaromatic compounds. Although the recent literature contains numerous examples of oxygenase-catalyzed nitro group displacements (33), little is known about the enzymes involved in these reactions. Monooxygenases that oxidize 4-and 2-nitrophenol (34, 36, 49) have been described, but only the 2-nitrophenol monooxygenase from Pseudomonas putida B2 has been purified and characterized (49). Recent evidence has suggested that some of the dioxygenases involved in the removal of nitro groups from aromatic compounds are multicomponent enzyme systems similar to the naphthalene dioxygenase enzyme system (2, 40). Burkholderia sp. strain DNT (formerly Pseudomonas sp. strain DNT) uses both mono-and dioxygenase enzymes to remove nitro groups (13,37,40) in an oxidative pathway that leads to the mineralization of 2,4-dinitrotoluene (2,4-DNT) (37).The initial attack on 2,4-DNT by...

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Section: Discussionmentioning

confidence: 99%

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Haigler¹,

Suen²,

Spain³

1996

J Bacteriol

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“…Analysis of the genomic sequence also revealed four new PKS gene clusters in S. chattanoogensis L10, including the scw PKS, scm PKS, PKSIa, and PKSIIc gene clusters. The scw PKS gene cluster contains homologous genes of all eight genes in whiE PKS gene clusters in several Streptomyces strains (33)(34)(35)(36)(37). The whiE PKS is a type II PKS, which is involved in structure-unknown spore pigment biosynthesis.…”

Section: Alignment Of Schppt and Schacps With Known Pptases Showed Thmentioning

confidence: 99%

Improvement of Natamycin Production by Engineering of Phosphopantetheinyl Transferases in Streptomyces chattanoogensis L10

Jiang

Wang

Ran

et al. 2013

Appl Environ Microbiol

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Phosphopantetheinyl transferases (PPTases) are essential to the activities of type I/II polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) through converting acyl carrier proteins (ACPs) in PKSs and peptidyl carrier proteins (PCPs) in NRPSs from inactive apo-forms into active holo-forms, leading to biosynthesis of polyketides and nonribosomal peptides. The industrial natamycin (NTM) producer,Streptomyces chattanoogensisL10, contains two PPTases (SchPPT and SchACPS) and five PKSs. Biochemical characterization of these two PPTases shows that SchPPT catalyzes the phosphopantetheinylation of ACPs in both type I PKSs and type II PKSs, SchACPS catalyzes the phosphopantetheinylation of ACPs in type II PKSs and fatty acid synthases (FASs), and the specificity of SchPPT is possibly controlled by its C terminus. Inactivation of SchPPT inS. chattanoogensisL10 abolished production of NTM but not the spore pigment, while overexpression of the SchPPT gene not only increased NTM production by about 40% but also accelerated productions of both NTM and the spore pigment. Thus, we elucidated a comprehensive phosphopantetheinylation network of PKSs and improved polyketide production by engineering the cognate PPTase in bacteria.

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“…[24] Moreover, the mec cluster displayed strikingly similarity in gene organization ( Figure 1) to the spore pigment biosynthetic gene clusters from Streptomyces collinus DSM 2012 (unpublished data), Streptomyces avermitilis MA-4680, [25] Streptomyces coelicolor, [26] and other streptomyces strains. [27][28][29] From these results it is reasonable to propose that the mec cluster is responsible for the biosynthesis of spore pigment in S. bottropensis.…”

Section: Resultsmentioning

confidence: 91%

Cloning and Heterologous Expression of Three Type II PKS Gene Clusters from Streptomyces bottropensis

Yan¹,

Probst²,

Linnenbrink³

et al. 2011

ChemBioChem

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Mensacarcin is a potent cytotoxic agent isolated from Streptomyces bottropensis. It possesses a high content of oxygen atoms and two epoxide groups, and shows cytostatic and cytotoxic activity comparable to that of doxorubicin, a widely used drug for antitumor therapy. Another natural compound, rishirilide A, was also isolated from the fermentation broth of S. bottropensis. Screening a cosmid library of S. bottropensis with minimal PKS-gene-specific primers revealed the presence of three different type II polyketide synthase (PKS) gene clusters in this strain: the msn cluster (mensacarcin biosynthesis), the rsl cluster (rishirilide biosynthesis), and the mec cluster (putative spore pigment biosynthesis). Interestingly, luciferase-like oxygenases, which are very rare in Streptomyces species, are enriched in both the msn cluster and the rsl cluster. Three cosmids, cos2 (containing the major part of the msn cluster), cos3 (harboring the mec cluster), and cos4 (spanning probably the whole rsl cluster) were introduced into the general heterologous host Streptomyces albus by intergeneric conjugation. Expression of cos2 and cos4 in S. albus led to the production of didesmethylmensacarcin (DDMM, a precursor of mensacarcin) and the production of rishirilide A and B (a precursor of rishirilide A), respectively. However, no product was detected from the expression of cos3. In addition, based on the results of isotope-feeding experiments in S. bottropensis, a putative biosynthesis pathway for mensacarcin is proposed.

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A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii

Cited by 23 publications

References 26 publications

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Purification and sequence analysis of 4-methyl-5-nitrocatechol oxygenase from Burkholderia sp. strain DNT

Improvement of Natamycin Production by Engineering of Phosphopantetheinyl Transferases in Streptomyces chattanoogensis L10

Cloning and Heterologous Expression of Three Type II PKS Gene Clusters from Streptomyces bottropensis

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