Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of
            <i>Escherichia coli</i>
            W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

Galán, Beatriz; Dı́az, Eduardo; Prieto, María Auxiliadora; Garcı́a, José Luis

doi:10.1128/jb.182.3.627-636.2000

Cited by 173 publications

(195 citation statements)

References 46 publications

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“…S1). Ksh1 activity can be eliminated either by deleting kshB1 or kshA1 (or both), but we decided to test the single deletion of kshB1 instead of kshA1 , because in the absence of the corresponding oxygenase component, the reductase component of multicomponent oxygenases usually acts as a gratuitous scavenger of reduction power, generating futile cycles that might reduce the efficiency of the biotransformation process (Galán et al ., 2000; Blank et al ., 2010) and therefore, in this sense, it can be more convenient to suppress the reductase instead the oxygenase component. However, it is also true that other reductase enzymes present in the cell could replace their function.…”

Section: Resultsmentioning

confidence: 99%

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

Galán

Uhía

García-Fernández

et al. 2016

Microbial Biotechnology

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SummaryA number of pharmaceutical steroid synthons are currently produced through the microbial side‐chain cleavage of natural sterols as an alternative to multi‐step chemical synthesis. Industrially, these synthons have been usually produced through fermentative processes using environmental isolated microorganisms or their conventional mutants. Mycobacterium smegmatis mc2155 is a model organism for tuberculosis studies which uses cholesterol as the sole carbon and energy source for growth, as other mycobacterial strains. Nevertheless, this property has not been exploited for the industrial production of steroidic synthons. Taking advantage of our knowledge on the cholesterol degradation pathway of M. smegmatis mc2155 we have demonstrated that the MSMEG_6039 (kshB1) and MSMEG_5941 (kstD1) genes encoding a reductase component of the 3‐ketosteroid 9α‐hydroxylase (KshAB) and a ketosteroid Δ1‐dehydrogenase (KstD), respectively, are indispensable enzymes for the central metabolism of cholesterol. Therefore, we have constructed a MSMEG_6039 (kshB1) gene deletion mutant of M. smegmatis MS6039 that transforms efficiently natural sterols (e.g. cholesterol and phytosterols) into 1,4‐androstadiene‐3,17‐dione. In addition, we have demonstrated that a double deletion mutant M. smegmatis MS6039‐5941 [ΔMSMEG_6039 (ΔkshB1) and ΔMSMEG_5941 (ΔkstD1)] transforms natural sterols into 4‐androstene‐3,17‐dione with high yields. These findings suggest that the catabolism of cholesterol in M. smegmatis mc2155 is easy to handle and equally efficient for sterol transformation than other industrial strains, paving the way for valuating this strain as a suitable industrial cell factory to develop à la carte metabolic engineering strategies for the industrial production of pharmaceutical steroids.

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Section: Resultsmentioning

confidence: 99%

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

Galán

Uhía

García-Fernández

et al. 2016

Microbial Biotechnology

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“…Although most flavin monooxygenases carry out flavin reduction and substrate oxidation in a single active site, two-component f lavindependent monooxygenases have been described that use a separate flavin reductase to catalyze the first step of the catalytic cycle (12,14). The reduced flavin is believed to diffuse into the active site of the monoxygenase component, because this catalytic cooperation does not appear to depend on protein-protein interactions (12)(13)(14). In analogy to these two-component flavin monooxygenases, RebF and RebH were proposed to form a two-component halogenase.…”

Section: Resultsmentioning

confidence: 99%

“…PrnA, the Trp-7-halogenase involved in the formation of pyrrolnitrin, is foremost among FADH 2 -dependent halogenases that have been described (9,10). In these studies, van Pée and coworkers (9,10) established the requirement for a separate flavin reductase to provide reduced FADH 2 for the halogenase, in analogy with two-component flavin monooxygenase systems (12)(13)(14). However, low in vitro activity of this system has precluded more detailed studies, so even a parameter as fundamental as catalytic efficiency has yet to be reported for a FADH 2 -dependent halogenase, and little is known about the mechanism of halogenation by these enzymes.…”

mentioning

confidence: 99%

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Yeh

Garneau²,

Walsh³

2005

Proc. Natl. Acad. Sci. U.S.A.

253

278

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The indolocarbazole antitumor agent rebeccamycin is modified by chlorine atoms on each of two indole moieties of the aglycone scaffold. These halogens are incorporated during the initial step of its biosynthesis from conversion of L-Trp to 7-chlorotryptophan. Two genes in the biosynthetic cluster, rebF and rebH, are predicted to encode the flavin reductase and halogenase components of an FADH 2-dependent halogenase, a class of enzymes involved in the biosynthesis of numerous halogenated natural products. Here, we report that, in the presence of O 2, chloride ion, and L-Trp as cosubstrates, purified RebH displays robust regiospecific halogenating activity to generate 7-chlorotryptophan over at least 50 catalytic cycles. Halogenation by RebH required the addition of RebF, which catalyzes the NADH-dependent reduction of FAD to provide FADH 2 for the halogenase. Maximal rates were achieved at a RebF͞RebH ratio of 3:1. In air-saturated solutions, a k cat of 1.4 min ؊1 was observed for the RebF͞RebH system but increased at least 10-fold in low-pO 2 conditions. RebH was also able to use bromide ions to generate monobrominated Trp. The demonstration of robust chlorinating activity by RebF͞RebH sets up this system for the probing of mechanistic questions regarding this intriguing class of enzymes.enzymology ͉ natural product biosynthesis ͉ flavoenzyme

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“…Whereas FADH 2 most commonly serves as a redox cofactor when it is tightly bound to enzymes, free FADH 2 is used as a co-substrate for monooxygenase reactions (42)(43)(44)(45)(46), including several from E. coli strains (28,47,48). Indirect evidence has suggested that FADH 2 may also assist in the activation of ribonucleotide reductase (33).…”

Section: Nadh Is Unlikely To Be the Direct Reductant Of Free Iron-mentioning

confidence: 99%

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

Woodmansee

Imlay

2002

Journal of Biological Chemistry

130

163

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When cells are exposed to external H 2 O 2 , the H 2 O 2 rapidly diffuses inside and oxidizes ferrous iron, thereby forming hydroxyl radicals that damage DNA. Thus the process of oxidative DNA damage requires only H 2 O 2 , free iron, and an as-yet unidentified electron donor that reduces ferric iron to the ferrous state. Previous work showed that H 2 O 2 kills Escherichia coli especially rapidly when respiration is inhibited either by cyanide or by genetic defects in respiratory enzymes. In this study we established that these respiratory blocks accelerate the rate of DNA damage. The respiratory blocks did not substantially affect the amounts of intracellular free iron or H 2 O 2 , indicating that that they accelerated damage because they increased the availability of the electron donor. The goal of this work was to identify that donor. As expected, the respiratory inhibitors caused a large increase in the amount of intracellular NADH. However, NADH itself was a poor reductant of free iron in vitro. This suggests that in non-respiring cells electrons are transferred from NADH to another carrier that directly reduces the iron. Genetic manipulations of the amounts of intracellular glutathione, NADPH, ␣-ketoacids, ferredoxin, and thioredoxin indicated that none of these was the direct electron donor. However, cells were protected from cyanide-stimulated DNA damage if they lacked flavin reductase, an enzyme that transfers electrons from NADH to free FAD. The K m value of this enzyme for NADH is much higher than the usual intracellular NADH concentration, which explains why its flux increased when NADH levels rose during respiratory inhibition. Flavins that were reduced by purified flavin reductase rapidly transferred electrons to free iron and drove a DNA-damaging Fenton system in vitro. Thus the rate of oxidative DNA damage can be limited by the rate at which electron donors reduce free iron, and reduced flavins become the predominant donors in E. coli when respiration is blocked. It remains unclear whether flavins or other reductants drive Fenton chemistry in respiring cells.

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Functional Analysis of the Small Component of the 4-Hydroxyphenylacetate 3-Monooxygenase of Escherichia coli W: a Prototype of a New Flavin:NAD(P)H Reductase Subfamily

Cited by 173 publications

References 46 publications

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

Mycobacterium smegmatis is a suitable cell factory for the production of steroidic synthons

Robust in vitro activity of RebF and RebH, a two-component reductase/halogenase, generating 7-chlorotryptophan during rebeccamycin biosynthesis

Reduced Flavins Promote Oxidative DNA Damage in Non-respiringEscherichia coli by Delivering Electrons to Intracellular Free Iron

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