A variety of Mycobacterium species contained the 5-deazaflavin coenzyme known as F 420 . Mycobacterium smegmatis was found to have a glucose-6-phosphate dehydrogenase that was dependent on F 420 as an electron acceptor and which did not utilize NAD or NADP. The enzyme was purified by ammonium sulfate fractionation, phenyl-Sepharose column chromatography, F 420 -ether-linked aminohexyl-Sepharose 4B affinity chromatography, and quaternary aminoethyl-Sephadex column chromatography, and the sequence of the first 26 Nterminal amino acids has been determined. The response of enzyme activity to a range of pHs revealed a two-peak pattern, with maxima at pH 5.5 and 8.0. The apparent K m values for F 420 and glucose-6-phosphate were, respectively, 0.004 and 1.6 mM. The apparent native and subunit molecular masses were 78,000 and ϳ40,000 Da, respectively.The electron transfer coenzyme known as F 420 , a 5-deazaflavin, is found in few bacteria. However, it is a major component in the energy metabolism pathways of methanogenic bacteria and Archaeoglobus fulgidus (5,11,18,19,21,29,32,33), where it participates in a variety of two-electron transfer reactions. It is also found in several Streptomyces species (13), where it is used in the synthesis of tetracyclines (41) and lincomycin (8,35), and in Streptomyces griseus, Scenedesmus acutus, Anacystis nidulans, and methanogens it is a component of the DNA repair enzyme photolyase (22,24,31,40). In Streptomyces species, Archaeoglobus fulgidus, and methanogens it exchanges electrons with NADP via the enzyme NADP-F 420 oxidoreductase (20,23,34,56). F 420 is present in Mycobacterium avium (44) and Mycobacterium tuberculosis (13), but in these organisms its metabolic role is unknown. Actually, long before the discovery of F 420 in methanogens, Cousins isolated a yellow pigment from Mycobacterium smegmatis which had a UV-visible spectrum very similar to that of F 420 (9) and Sutton reported on an NADP-like electron transfer component in Mycobacterium phlei (52, 53) which is likely to have been F 420 .Chemical and biochemical properties of F 420 are more similar to those of nicotinamides than to those of flavins, despite its structural resemblance to the latter. Its reduced form is stable for hours in air, it participates in most instances in two-electron transfer reactions, it has a redox potential of Ϫ350 mV, and it reacts rapidly with flavins (29, 54). Figure 1 shows the structure of the monoglutamate form of F 420 found in M. avium (44), but methanogens and others may contain multiglutamate residues.Because of increasing problems in treatment of human disease caused by M. tuberculosis and M. avium infections arising from the global AIDS epidemic, faltering public health systems, and increasing cases of multiple drug resistance in tuberculosis patients (7,25,26,42), we have become interested in examining F 420 -dependent reactions in mycobacteria. Currently, the role of F 420 in mycobacteria and its distribution among different species or strains are unknown. In this study we examine...

In mycobacteria, F420, a deazaflavin derivative, acts as a hydride transfer coenzyme for an F 420-specific glucose-6-phosphate dehydrogenase (Fgd). Physiologically relevant reactions in the mycobacteria that use Fgd-generated reduced F 420 (F 420 H 2 ) are unknown. In this work, F420H2 was found to be oxidized by NO only in the presence of oxygen. Further analysis demonstrated that NO 2, produced from NO and O 2 , was the oxidant. UV-visible spectroscopic and NO-sensor-based analyses proved that F420H2 reduced NO 2 to NO. This reaction could serve as a defense system for Mycobacterium tuberculosis, which is more sensitive to NO 2 than NO under aerobic conditions. Activated macrophages produce NO, which in acidified phagosomes is converted to NO 2. Hence, by converting NO 2 back to NO with F420H2, M. tuberculosis could decrease the effectiveness of antibacterial action of macrophages; such defense would correspond to active tuberculosis conditions where the bacterium grows aerobically. This hypothesis was consistent with the observation that a mutant strain of Mycobacterium smegmatis, a nonpathogenic relative of M. tuberculosis, which either did not produce or could not reduce F 420, was Ϸ4-fold more sensitive to NO 2 than the wild-type strain. The phenomenon is reminiscent of the anticancer activity of ␥-tocopherol, which reduces NO 2 to NO and protects human cells from NO2-induced carcinogenesis.macrophage ͉ deazaflavin ͉ nitrogen dioxide ͉ nitric oxide ͉ tuberculosis C oenzyme F 420 , a 7,8-didemethyl-8-hydroxy-5-deazaflavin derivative ( Fig. 1), is a 2-electron or hydride transfer restricted redox catalyst (E o Ј ϭ Ϫ360 mV) similar to the nicotinamides (E o Ј ϭ Ϫ320 mV) (1, 2). F 420 is found in all methanogenic and certain nonmethanogenic archaea, where it participates in energy metabolism, NADP reduction, oxygen detoxification, and sulfite reduction (3-6). In the bacterial domain, F 420 is found in certain members of the Actinobacteria phylum, such as Mycobacterium species (7). These organisms express an F 420 -dependent glucose-6-phosphate dehydrogenase (Fgd, Reaction 1) (7, 8).Glucose 6-phosphate ϩ F 420 3 6-phosphogluconate ϩ F 420 H 2 [Reaction 1]The physiological fate of F 420 H 2 produced by Fgd is unknown. An insertional inactivation of fbiC, an essential gene for the synthesis of the deazaflavin chromophore or catalytic unit of F 420 (9), renders Mycobacterium tuberculosis hypersusceptible to acidified nitrite (10). This in vitro treatment simulates an environment inside the phagosomes of an infected-activated macrophage, which produces nitric oxide (NO) by the action of inducible nitric oxide synthase (iNOS or NOS2) (11). Upon acidification of a phagosome, nitrite, a major product of NO oxidation, is converted to nitrous acid (HNO 2 ; pK a ϭ 3.16) (12), which in turn dismutates to NO and NO 2 (10, 13); NO 2 arises also from a reaction of NO with O 2 (14). These observations suggest that the pathogenic mycobacteria could use F 420 H 2 to combat an attack of reactive nitrogen intermediates generated ...

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Glucose 6-phosphate (G6P) is a metabolic intermediate with many possible cellular fates. In mycobacteria, G6P is a substrate for an enzyme, F 420 -dependent glucose-6-phosphate dehydrogenase (Fgd), found in few bacterial genera. Intracellular G6P levels in six Mycobacterium sp. were remarkably higher (ϳ17-130-fold) than Escherichia coli and Bacillus megaterium. The high G6P level in Mycobacterium smegmatis may result from 10 -25-fold higher activity of the gluconeogenic enzyme fructose-1,6-bisphosphatase when grown on glucose, glycerol, or acetate compared with B. megaterium and E. coli. In M. smegmatis this coincided with up-regulation of the first gluconeogenic enzyme, phosphoenolpyruvate carboxykinase, when acetate was the carbon source, suggesting a cellular program for maintaining high G6P levels. G6P was depleted in cells under oxidative stress induced by redox cycling agents plumbagin and menadione, whereas an fgd mutant of M. smegmatis used G6P less well under such conditions. The fgd mutant was more sensitive to these agents and, in contrast to wild type, was defective in its ability to reduce extracellular plumbagin and menadione. These data suggest that intracellular G6P in mycobacteria serves as a source of reducing power and, with the mycobacteriaspecific Fgd-F 420 system, plays a protective role against oxidative stress.

The ability of Mycobacterium tuberculosis to manipulate and evade human immune system is in part due to its extraordinarily complex cell wall. One of the key components of this cell wall is a family of lipids called mycolic acids. Oxygenation of mycolic acids generating methoxy- and ketomycolic acids enhances the pathogenic attributes of M. tuberculosis. Thus, the respective enzymes are of interest in the research on mycobacteria. The generation of methoxy- and ketomycolic acids proceeds through intermediary formation of hydroxymycolic acids. While the methyl transferase that generates methoxymycolic acids from hydroxymycolic acids is known, hydroxymycolic acids dehydrogenase that oxidizes hydroxymycolic acids to ketomycolic acids has been elusive. We found that hydroxymycolic acid dehydrogenase is encoded by the rv0132c gene and the enzyme utilizes F420, a deazaflavin coenzyme, as electron carrier, and accordingly we called it F420-dependent hydroxymycolic acid dehydrogenase. This is the first report on the involvement of F420 in the synthesis of a mycobacterial cell envelope. Also, F420-dependent hydroxymycolic acid dehydrogenase was inhibited by PA-824, and therefore, it is a previously unknown target for this new tuberculosis drug.