MSMEG_3955 from Mycobacterium smegmatis is a FMN bounded homotrimeric NAD(P)H:Flavin mononucleotide (FMN) oxidoreductase

Abstract

Background: Tuberculosis (TB) remains an important public health problem since it is the major cause of elevated morbidity and mortality globally. Previous works have shown that Mycobacterium tuberculosis (Mtb); the prime causative agent of the deadly disease has dormancy survival regulator (DosR) regulon, a two-component regulatory system which controls the transcription of more than 50 genes. However, the structure and detailed functions of these DosR regulated genes are largely undetermined. Out of many DosR regulon genes, Rv3131 gets up regulated in hypoxic conditions and was believed to encode for a nitroreductase flavoprotein. The utilization of mycobacteria-specific model systems has greatly added to our understanding of the molecular mechanisms involved in the life cycle and pathogenesis of Mtb.

Results: In this study the non-pathogenic mycobacterial model organism Mycobacterium smegmatis (Msmeg) was used to reveal the structure and function of MSMEG_3955; which is a homologue of Rv3131 from Mtb. Using chromatography and spectroscopy techniques it was revealed that cofactor flavin mononucleotide (FMN) was bound to flavoprotein MSMEG_3955. Consistent with the homology modelling predictions, Circular Dichroism (CD) analysis indicated that the MSMEG_3955 is composed of 39.3% α-helix and 24.9% β-pleated sheets. In contrast to the current notions, the enzymatic assays performed in the present study revealed that MSMEG_3955 was not capable of reducing nitro substrates but showed NADPH dependent FMN oxidoreductase activity. Also, gel permeation chromatography, dynamic light scattering and native acidic gels showed that MSMEG_3955 exists as a homotrimer. Furthermore, the presence of NADPH dependent FMN oxidoreductase and homotrimeric existence could be an alternative function of the protein to help the bacteria survive in dormant state or may be involved in other biochemical pathways.

Conclusion: MSMEG_3955 is a FMN bound flavoprotein, which exits as a trimer under in vitro conditions. There is no disulphide linkages in between the three protomers of the homotrimer MSMEG_3955. It has a NADPH dependent FMN oxidoreductase activity.

Fig. 1

Electrophoretic gel pictures. a Gel picture of amplified gene MSMEG_3955 from genomic DNA. b Gel picture of double digested clone of pET 28a vector containing gene MSMEG_3955. c Gel picture of 12% acidic SDS-PAGE of expressed reduced purified recombinant protein, expressed reduced crude protein along with controls

Fig. 2

Polyacrylamide Gel Electrophoresis for analysis of the presence or absence of disulphide linkage within the homotrimeric protein. a Gel picture of wild-type and cys-63 mutated native protein MSMEG_3955 run on 12% NATIVE-PAGE. b Non-reduced protein MSMEG_3955 run on 12% SDS-PAGE

Fig. 3

Oligomeric state analysis of protein MSMEG_3955. a Gel picture of native protein run on 10% NATIVE-PAGE. b Percentage particle size distribution graph of protein showing diameter of molecule ~ 7.8 nm, analysed by DLS. c Gel permeation chromatography showing elution profile of protein (~ 115 kDa) with BSA (132 kDa) and Lysozyme (14 kDa) as standards

Fig. 4

Secondary structure analysis of MSMEG_3955. a Graphical representation of Circular Dichroism spectrum of MSMEG_3955 protein (8 μM) in 10 mM HEPES. b Modelled structure of MSMEG_3955, Helices are colored blue, sheets are red and coils are purple

Fig. 5

Analysis of cofactor bound to the protein MSMEG_3955. a The cofactor separated from protein MSMEG_3955 migrates along with FMN (control Lane-1) on thin layer chromatography (TLC). b UV Spectrum showing maximum absorbance at 446 nm wavelength for cofactor detached from protein treated with 10% SDS

Fig. 6

Measurement of FMN reductase activity using NADPH as an electron donor. a The protein/enzyme MSMEG_3955 showed oxidative NADPH dependent FMN reductase activity in contrast to control. b The selected region of ¹H NMR spectra of FMN in oxidized (red) and reduced (blue) form recorded at 500 MHz, pH -7.6. The Flavin ring proton is represented by letter “a”