Purification of a novel coenzyme F-420-dependent glucose-6-phosphate dehydrogenase from Mycobacterium smegmatis

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 N-terminal 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 similar to 40,000 Da, respectively.