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Summary: Deletion of gene Rv3676 in Mycobacterium tuberculosis coding for a transcription factor belonging to the cAMP receptor protein (CRP) family caused growth defects in laboratory medium, in bone marrow‐derived macrophages and in a mouse model of tuberculosis. Transcript profiling of M. tuberculosis grown in vitro identified 16 genes with significantly altered expression in the mutant compared with the wild type. Analysis of the DNA sequences upstream of the corresponding open reading frames revealed that 12 possessed sequences related to a consensus CRP binding site that could represent the sites of action of Rv3676. These included rpfA, lprQ, whiB1 and ahpC among genes with enhanced expression in the wild type, and Rv3616c‐Rv3613c, Rv0188 and lipQ among genes exhibiting enhanced expression in the mutant. The activity of an rpfA::lacZ promoter fusion was lowered in the Rv3676 mutant and by mutation of the predicted Rv3676 binding site. Moreover, the product of Rv3676 (isolated as a TrxA fusion protein) interacted specifically with the rpfA promoter, and binding was inhibited by mutation of the Rv3676 site. Although Rv3676 retains four of the six amino acid residues that bind cAMP in Escherichia coli CRP addition of cAMP did not enhance Rv3676 binding at the rpfA promoter in vitro. In summary, it has been shown that Rv3676 is a direct regulator of rpfA expression, and because rpfA codes for a resuscitation promoting factor this may implicate Rv3676 in reactivation of dormant M. tuberculosis infections.

SUMMARY: 5-Deazaflavin cofactors enhance the metabolic flexibility of microorganisms by catalyzing a wide range of challenging enzymatic redox reactions. While structurally similar to riboflavin, 5-deazaflavins have distinctive and biologically useful electrochemical and photochemical properties as a result of the substitution of N-5 of the isoalloxazine ring for a carbon. 8-Hydroxy-5-deazaflavin (F_o) appears to be used for a single function: as a light-harvesting chromophore for DNA photolyases across the three domains of life. In contrast, its oligoglutamyl derivative F₄₂₀is a taxonomically restricted but functionally versatile cofactor that facilitates many low-potential two-electron redox reactions. It serves as an essential catabolic cofactor in methanogenic, sulfate-reducing, and likely methanotrophic archaea. It also transforms a wide range of exogenous substrates and endogenous metabolites in aerobic actinobacteria, for example mycobacteria and streptomycetes. In this review, we discuss the physiological roles of F₄₂₀in microorganisms and the biochemistry of the various oxidoreductases that mediate these roles. Particular focus is placed on the central roles of F₄₂₀in methanogenic archaea in processes such as substrate oxidation, C₁pathways, respiration, and oxygen detoxification. We also describe how two F₄₂₀-dependent oxidoreductase superfamilies mediate many environmentally and medically important reactions in bacteria, including biosynthesis of tetracycline and pyrrolobenzodiazepine antibiotics by streptomycetes, activation of the prodrugs pretomanid and delamanid byMycobacterium tuberculosis, and degradation of environmental contaminants such as picrate, aflatoxin, and malachite green. The biosynthesis pathways of F_oand F₄₂₀are also detailed. We conclude by considering opportunities to exploit deazaflavin-dependent processes in tuberculosis treatment, methane mitigation, bioremediation, and industrial biocatalysis.

Summary: Aflatoxins are polyaromatic mycotoxins that contaminate a range of food crops as a result of fungal growth and contribute to serious health problems in the developing world because of their toxicity and mutagenicity. Although relatively resistant to biotic degradation, aflatoxins can be metabolized by certain species of Actinomycetales. However, the enzymatic basis for their breakdown has not been reported until now. We have identified nine Mycobacterium smegmatis enzymes that utilize the deazaflavin cofactor F₄₂₀H₂ to catalyse the reduction of the α,β‐unsaturated ester moiety of aflatoxins, activating the molecules for spontaneous hydrolysis and detoxification. These enzymes belong to two previously uncharacterized F₄₂₀H₂ dependent reductase (FDR‐A and ‐B) families that are distantly related to the flavin mononucleotide (FMN) dependent pyridoxamine 5′‐phosphate oxidases (PNPOxs). We have solved crystal structures of an enzyme from each FDR family and show that they, like the PNPOxs, adopt a split barrel protein fold, although the FDRs also possess an extended and highly charged F₄₂₀H₂ binding groove. A general role for these enzymes in xenobiotic metabolism is discussed, including the observation that the nitro‐reductase Rv3547 from Mycobacterium tuberculosis that is responsible for the activation of bicyclic nitroimidazole prodrugs belongs to the FDR‐A family.