Type I flavohemoglobin of mycobacterium smegmatis is a functional nitric oxide dioxygenase

Thakur, Naveen; Gupta, Sanjay; Hade, Mangesh Dattu; Dikshit, Kanak L.

doi:10.1002/iub.1275

Cited by 7 publications

(4 citation statements)

References 30 publications

(54 reference statements)

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“…These observations provide not only insights into the metabolism of (alkyl)sulfur-bearing benzimidazole xenobiotics of clinical use, such as ABZ and fenbendazole, but raise the possibility that other organisms harboring superoxide-producing enzymes are able to metabolize ABZ in this way. This, in turn, is consistent with the observation that gFlHb, like other flavohemoglobins, is a multi-substrate enzyme [ 34 , 90 ]⁠.…”

Section: Discussionsupporting

confidence: 91%

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

et al. 2022

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Giardia duodenalis causes giardiasis, a major diarrheal disease in humans worldwide whose treatment relies mainly on metronidazole (MTZ) and albendazole (ABZ). The emergence of ABZ resistance in this parasite has prompted studies to elucidate the molecular mechanisms underlying this phenomenon. G. duodenalis trophozoites convert ABZ into its sulfoxide (ABZSO) and sulfone (ABZSOO) forms, despite lacking canonical enzymes involved in these processes, such as cytochrome P450s (CYP450s) and flavin-containing monooxygenases (FMOs). This study aims to identify the enzyme responsible for ABZ metabolism and its role in ABZ resistance in G. duodenalis. We first determined that the iron-containing cofactor heme induces higher mRNA expression levels of flavohemoglobin (gFlHb) in Giardia trophozoites. Molecular docking analyses predict favorable interactions of gFlHb with ABZ, ABZSO and ABZSOO. Spectral analyses of recombinant gFlHb in the presence of ABZ, ABZSO and ABZSOO showed high affinities for each of these compounds with Kd values of 22.7, 19.1 and 23.8 nM respectively. ABZ and ABZSO enhanced gFlHb NADH oxidase activity (turnover number 14.5 min-1), whereas LC-MS/MS analyses of the reaction products showed that gFlHb slowly oxygenates ABZ into ABZSO at a much lower rate (turnover number 0.01 min-1). Further spectroscopic analyses showed that ABZ is indirectly oxidized to ABZSO by superoxide generated from the NADH oxidase activity of gFlHb. In a similar manner, the superoxide-generating enzyme xanthine oxidase was able to produce ABZSO in the presence of xanthine and ABZ. Interestingly, we find that gFlHb mRNA expression is lower in albendazole-resistant clones compared to those that are sensitive to this drug. Furthermore, all albendazole-resistant clones transfected to overexpress gFlHb displayed higher susceptibility to the drug than the parent clones. Collectively these findings indicate a role for gFlHb in ABZ conversion to its sulfoxide and that gFlHb down-regulation acts as a passive pharmacokinetic mechanism of resistance in this parasite.

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Section: Discussionsupporting

confidence: 91%

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

et al. 2022

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“…Mtb FHb and Ms FHbII lack a NADH binding site and their NADH oxidase activity is attenuated ( Gupta et al., 2012 ; Thakur et al., 2018 ) unlike conventional type I FHb ( Ms FHbI) of M. smegmatis ( Thakur et al., 2014 ) and other bacteria. Since thioredoxin reductases are NADPH-dependent oxidoreductases and Mtb FHb and Ms FHbII carry thioredoxin reductase-like FAD binding motif, we checked whether these FHbs are able to oxidize NADPH.…”

Section: Resultsmentioning

confidence: 99%

“…The assay was performed in 50 mM phosphate buffer (pH 7.0) containing 1 mM protein ( Mtb FHb or Ms FHbII) and 1 mM EDTA. Reaction was initiated by adding 250 μM NADH or NADPH and spectra were recorded at 340 nm at specified time intervals as mentioned previously ( Thakur et al., 2014 ).…”

Section: Methodsmentioning

confidence: 99%

New Insights Into the Function of Flavohemoglobin in Mycobacterium tuberculosis: Role as a NADPH-Dependent Disulfide Reductase and D-Lactate-Dependent Mycothione Reductase

Thakur

Sharma

Hade

et al. 2022

Front. Cell. Infect. Microbiol.

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Mycobacterium tuberculosis (Mtb) produces an unconventional flavohemoglobin (MtbFHb) that carries a FAD-binding site similar to D-lactate dehydrogenases (D-LDH) and oxidizes D-lactate into pyruvate. The molecular mechanism by which MtbFHb functions in Mtb remains unknown. We discovered that the D-LDH-type FAD-binding site in MtbFHb overlaps with another FAD-binding motif similar to thioredoxin reductases and reduces DTNB in the presence of NADPH similar to trxB of Mtb. These results suggested that MtbFHb is functioning as a disulfide oxidoreductase. Interestingly, D-lactate created a conformational change in MtbFHb and attenuated its ability to oxidize NADPH. Mass spectroscopy demonstrated that MtbFHb reduces des-myo-inositol mycothiol in the presence of D-lactate unlike NADPH, indicating that D-lactate changes the specificity of MtbFHb from di-thiol to di-mycothiol. When M. smegmatis carrying deletion in the fhbII gene (encoding a homolog of MtbFHb) was complemented with the fhb gene of Mtb, it exhibited four- to fivefold reductions in lipid peroxidation and significant enhancement in the cell survival under oxidative stress. These results were corroborated by reduced lipid peroxidation and enhanced cell survival of wild-type M. smegmatis after overexpression of the fhb gene of Mtb. Since D-lactate is a by-product of lipid peroxidation and MtbFHb is a membrane-associated protein, D-lactate-mediated reduction of mycothiol disulfide by MtbFHb may uniquely equip Mtb to relieve the toxicity of D-lactate accumulation and protect the cell from oxidative damage, simultaneously balancing the redox environment under oxidative stress that may be vital for the pathogenesis of Mtb.

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“…In previous studies, Rrf2 family transcription factors controlled the expression of genes in a wide range of bacteria in response to nitric oxide, 36 iron homeostasis, 37 toxic oxidants, 38 nitric oxide, and nitrosative stress. 39 The ASCH domain-containing protein is a component of a putative RNA-interacting protein complex, and it is likely to function as an RNA-binding domain in environments related to RNA processing, coactivation, and possibly, prokaryotic translation regulation. 40,41 We speculated that the sp1764, as a regulator, was the main factor causing the change in butenyl-spinosyn biosynthesis.…”

Section: Deletion Of Clu13mentioning

confidence: 99%

Flaviolin-Like Gene Cluster Deletion Optimized the Butenyl-Spinosyn Biosynthesis Route inSaccharopolyspora pogona

Tang

Chen

et al. 2021

ACS Synth. Biol.

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Reduction and optimization of the microbial genome is an important strategy for constructing synthetic biological chassis cells and overcoming obstacles in natural product discovery and production. However, it is of great challenge to discover target genes that can be deleted and optimized due to the complicated genome of actinomycetes. Saccharopolyspora pogona can produce butenyl-spinosyn during aerobic fermentation, and its genome contains 32 different gene clusters. This suggests that there is a large amount of potential competitive metabolism in S. pogona, which affects the biosynthesis of butenyl-spinosyn. By analyzing the genome of S. pogona, six polyketide gene clusters were identified. From those, the complete deletion of clu13, a flaviolin-like gene cluster, generated a high butenyl-spinosyn-producing strain. Production of this strain was 4.06-fold higher than that of the wildtype strain. Transcriptome profiling revealed that butenyl-spinosyn biosynthesis was not primarily induced by the polyketide synthase RppA-like but was related to hypothetical protein Sp1764. However, the repression of sp1764 was not enough to explain the enormous enhancement of butenyl-spinosyn yields in S. pogona-Δclu13. After the comparative proteomic analysis of S. pogona-Δclu13 and S. pogona, two proteins, biotin carboxyl carrier protein (BccA) and response regulator (Reg), were investigated, whose overexpression led to great advantages of butenyl-spinosyn biosynthesis. In this way, we successfully discovered three key genes that obviously optimize the biosynthesis of butenyl-spinosyn. Gene cluster simplification performed in conjunction with multiomics analysis is of great practical significance for screening dominant chassis strains and optimizing secondary metabolism. This work provided an idea about screening key factors and efficient construction of production strains.

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Type I flavohemoglobin of mycobacterium smegmatis is a functional nitric oxide dioxygenase

Cited by 7 publications

References 30 publications

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

Giardia duodenalis: Flavohemoglobin is involved in drug biotransformation and resistance to albendazole

New Insights Into the Function of Flavohemoglobin in Mycobacterium tuberculosis: Role as a NADPH-Dependent Disulfide Reductase and D-Lactate-Dependent Mycothione Reductase

Flaviolin-Like Gene Cluster Deletion Optimized the Butenyl-Spinosyn Biosynthesis Route inSaccharopolyspora pogona

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