A new Gateway® vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

Goldstone, Rachael M.; Moreland, Nicole J.; Bashiri, Ghader; Baker, Edward N.; Lott, J.S.

doi:10.1016/j.pep.2007.08.015

Cited by 47 publications

(63 citation statements)

References 11 publications

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“…M. smegmatis FGD, which facilitates the formation of reduced F420, has a higher KM for glucose-6-phosphate than the enzyme from M. tuberculosis (1.6 mM vs. 0.1 mM, respectively) (Bashiri et al, 2008). The combination of potential differences in the tertiary structure of Ddn and/or lower formation of reduced F420 may explain the lack of production of the des-nitro metabolite ('1') in M. smegmatis, and hence, poor activity of PA-824 in this species.…”

Section: Figurementioning

confidence: 91%

“…Positive clones were selected on lysogeny broth (LB) agar plates containing ampicillin (100 mg·mL -1 ) and restriction digestion with BsrGI was used to select the correct size inserts. pDESTsmg positive clones were selected on low salt LB agar plates (pH 8.0) containing hygromycin B (50 mg·mL -1 ) (Goldstone et al, 2008). E. coli BL21pRP cells were used to express His-(pDEST17), GST-(pDEST15) and MBP-Ddn (pDEST566) constructs.…”

Section: Ddn Cloning Expression and Purificationmentioning

confidence: 99%

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Comparative bioactivation of the novel anti‐tuberculosis agent PA‐824 in Mycobacteria and a subcellular fraction of human liver

Dogra

Bashiri

et al. 2010

British J Pharmacology

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BACKGROUND AND PURPOSE PA‐824 is a 2‐nitroimidazooxazine prodrug currently in Phase II clinical trial for tuberculosis therapy. It is bioactivated by a deazaflavin (F420)‐dependent nitroreductase (Ddn) isolated from Mycobacterium tuberculosis to form a des‐nitro metabolite. This releases toxic reactive nitrogen species which may be responsible for its anti‐mycobacterial activity. There are no published reports of mammalian enzymes bioactivating this prodrug. We have investigated the metabolism of PA‐824 following incubation with a subcellular fraction of human liver, in comparison with purified Ddn, M. tuberculosis and Mycobacterium smegmatis.EXPERIMENTAL APPROACH PA‐824 (250 µM) was incubated with the 9000×g supernatant (S9) of human liver homogenates, purified Ddn, M. tuberculosis and M. smegmatis for metabolite identification by liquid chromatography mass spectrometry analysis.KEY RESULTS PA‐824 was metabolized to seven products by Ddn and M. tuberculosis, with the major metabolite being the des‐nitro product. Six of these products, but not the des‐nitro metabolite, were also detected in M. smegmatis. In contrast, only four of these metabolites were observed in human liver S9; M3, a reduction product previously proposed as an intermediate in the Ddn‐catalyzed des‐nitrification and radiolytic reduction of PA‐824; two unidentified metabolites, M1 and M4, which were products of M3; and a haem‐catalyzed product of imidazole ring hydration (M2).CONCLUSIONS AND IMPLICATIONS PA‐824 was metabolized by des‐nitrification in Ddn and M. tuberculosis, but this does not occur in human liver S9 and M. smegmatis. Thus, PA‐824 was selectively bioactivated in M. tuberculosis and there was no evidence for ‘cross‐activation’ by human enzymes.

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Section: Figurementioning

confidence: 91%

Section: Ddn Cloning Expression and Purificationmentioning

confidence: 99%

Comparative bioactivation of the novel anti‐tuberculosis agent PA‐824 in Mycobacteria and a subcellular fraction of human liver

Dogra

Bashiri

et al. 2010

British J Pharmacology

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“…Positive entry clones were selected on LB agar medium supplemented with 50 g/ml kanamycin and were then verified using BsrGI digestion and sequencing. The resulting entry clones were used to clone the full-length construct into pDEST17 (21) and pDESTsmg (22) vectors using an LR reaction. The expression construct for pDEST17 was selected on LB agar plates containing 100 g/ml ampicillin.…”

Section: Pcr Amplification and Cloning-mentioning

confidence: 99%

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

Bashiri

Rehan

Sreebhavan

et al. 2016

Journal of Biological Chemistry

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Cofactor F 420 is an electron carrier with a major role in the oxidoreductive reactions of Mycobacterium tuberculosis, the causative agent of tuberculosis. A ␥-glutamyl ligase catalyzes the final steps of the F 420 biosynthesis pathway by successive additions of L-glutamate residues to F 420 -0, producing a poly-␥-glutamate tail. The enzyme responsible for this reaction in archaea (CofE) comprises a single domain and produces F 420 -2 as the major species. The homologous M. tuberculosis enzyme, FbiB, is a two-domain protein and produces F 420 with predominantly 5-7 L-glutamate residues in the poly-␥-glutamate tail. The N-terminal domain of FbiB is homologous to CofE with an annotated ␥-glutamyl ligase activity, whereas the C-terminal domain has sequence similarity to an FMN-dependent family of nitroreductase enzymes. Here we demonstrate that full-length FbiB adds multiple L-glutamate residues to F 420 -0 in vitro to produce F 420 -5 after 24 h; communication between the two domains is critical for full ␥-glutamyl ligase activity. We also present crystal structures of the C-terminal domain of FbiB in apo-, F 420 -0-, and FMN-bound states, displaying distinct sites for F 420 -0 and FMN ligands that partially overlap. Finally, we discuss the features of a full-length structural model produced by small angle x-ray scattering and its implications for the role of N-and C-terminal domains in catalysis.The cofactor F 420 is a flavin derivative that is sporadically distributed among microorganisms, mainly archaea and actinobacteria (including mycobacteria). F 420 has been emerging as a new player in the biology of mycobacteria (1), with increasing numbers of F 420 -utilizing proteins characterized from different mycobacterial species (2-8). This cofactor has been suggested to protect Mycobacterium tuberculosis, the causative agent of tuberculosis, against oxidative and nitrosative stress during pathogenesis (9 -11). At the biochemical level, cofactor F 420 functions as a hydride transfer agent in oxidoreductive reactions with a lower redox potential than that of NAD(P) ϩ (12). The biosynthesis pathway of cofactor F 420 has been investigated in both archaeal and mycobacterial species. In the current view of the proposed pathway, the first intermediate with the complete chromophore (7, ) is produced by FO synthase (FbiC in mycobacteria (13) and CofGH in archaea (14)). A transferase enzyme (FbiA in mycobacteria (15) and CofD in archaea (16)) subsequently catalyzes the addition of a 2-phospho-L-lactate moiety to FO to produce F 420 -0 (F 420 with no poly-␥-glutamate tail). The final step of the pathway is performed by a ␥-glutamyl ligase (FbiB in mycobacteria (15) and CofE in archaea (17)) that catalyzes successive additions of L-glutamate residues to F 420 -0 (Fig. 1A).The length of the poly-␥-glutamate tail varies between archaeal and mycobacterial species; in archaea, two L-glutamate residues are seen (18), whereas in mycobacteria, up to nine residues are present (3, 19). There exists an intriguing difference between the ...

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“…It incorporates a higher proportion of glycine, alanine, proline and arginine and Mtb post-translational modification machinery is lacking in E. coli (35,36). M. smegmatis, a fast-growing saprophytic relative of Mtb, is a potential expression host for Mtb genes overcoming many of the restrictions of E. coli (36)(37)(38)(39). Unexpectedly, however, only nine proteins of H37Rv represented by 27 structures were produced in M. smegmatis (37,(40)(41)(42)(43)(44)(45)(46).…”

Section: Technical Aspects Of Mtb Structural Analysismentioning

confidence: 99%

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

et al. 2015

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Tuberculosis threatens human health nowhere more than in developing countries with large malnourished and/or immune-compromised (e.g. HIV infected) populations. The etiological agent, Mycobacterium tuberculosis (Mtb), is highly infectious and current interventions demonstrate limited ability to control the epidemic in particular of drug resistant Mtb strains. New drugs and vaccines are thus urgently required. Structural biologists are critical to the TB research community. By identifying potential drug targets and solving their three dimensional structures they open new avenues of identifying potential inhibitors complementing the screening of novel compounds and the investigation of Mtb's molecular physiology by pharmaceutical companies and academic researchers. Much effort has gone into structurally elucidating the Mtb proteome though much remains to be done with progress primarily limited by technological constraints. We review the currently available data for Mtb H37Rv to extract the lessons they have taught us. KeywordsMycobacterium tuberculosis, structural genomics, drug target AbbreviationsAll abbreviations in the manuscript are standard in the field.

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A new Gateway® vector and expression protocol for fast and efficient recombinant protein expression in Mycobacterium smegmatis

Cited by 47 publications

References 11 publications

Comparative bioactivation of the novel anti‐tuberculosis agent PA‐824 in Mycobacteria and a subcellular fraction of human liver

Comparative bioactivation of the novel anti‐tuberculosis agent PA‐824 in Mycobacteria and a subcellular fraction of human liver

Elongation of the Poly-γ-glutamate Tail of F420 Requires Both Domains of the F420:γ-Glutamyl Ligase (FbiB) of Mycobacterium tuberculosis

Assessing the progress of Mycobacterium tuberculosis H37Rv structural genomics

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