F(420)-dependent secondary alcohol dehydrogenase (Adf) from methanogenic archaea is a member of the growing bacterial luciferase family which are all TIM barrel enzymes, most of which with an unusual nonprolyl cis peptide bond. We report here on the crystal structure of Adf from Methanoculleus thermophilicus at 1.8 A resolution in complex with a F(420)-acetone adduct. The knowledge of the F(420) binding mode in Adf provides the molecular basis for modeling F(420) and FMN into the other enzymes of the family. A nonprolyl cis peptide bond was identified as an essential part of a bulge that serves as backstop at the Re-face of F(420) to keep it in a bent conformation. The acetone moiety of the F(420)-acetone adduct is positioned at the Si-face of F(420) deeply buried inside the protein. Isopropanol can be reliably modeled and a hydrogen transfer mechanism postulated. His39 and Glu108 can be identified as key players for binding of the acetone or isopropanol oxygens and for catalysis.

Methanogenic archaea growing on ethanol or isopropanol as the electron donor for CO2 reduction to CH4 contain either an NADP-dependent or a coenzyme F420-dependent alcohol dehydrogenase. We report here that in both groups of methanogens, the N5, N10-methylenetetrahydromethanopterin dehydrogenase and the N5, N10-methylenetetrahydromethanopterin reductase, two enzymes involved in CO2 reduction to CH4, are specific for F420. This raised the question how F420H2 is regenerated in the methanogens with an NADP-dependent alcohol dehydrogenase. We found that these organisms contain catabolic activities of an enzyme catalyzing the reduction of F420 with NADPH. The F420-dependent NADP reductase from Methanogenium organophilum was purified and characterized. The N-terminal amino acid sequence showed 42% sequence identity to a putative gene product in Methanococcus jannaschii, the total genome of which has recently been sequenced.

The unfolded states of three homologous proteins with a very similar fold have been investigated by heteronuclear NMR spectroscopy. Secondary structure propensities as derived from interpretation of chemical shifts and motional restrictions as evidenced by heteronuclear 15 N relaxation rates have been analyzed in the reduced unfolded states of hen lysozyme and the calcium-binding proteins bovine a-lactalbumin and human a-lactalbumin. For all three proteins, significant deviations from random-coil predictions can be identified; in addition, the unfolded states also differ from each other, despite the fact that they possess very similar structures in their native states. Deviations from random-coil motional properties are observed in the a-and the b-domain in bovine a-lactalbumin and lysozyme, while only regions within the a-domain deviate in human a-lactalbumin. The motional restrictions and residual secondary structure are determined both by the amino acid sequence of the protein and by residual longrange interactions. Even a conservative single point mutation from I to L in a highly conserved region between the two a-lactalbumins results in considerable differences in the motional properties. Given the differences in oxidative folding between hen lysozyme and a-lactalbumin, the results obtained on the unfolded states suggest that residual long-range interactions, i.e., those between the a-and the b-domain of lysozyme, may act as nucleation sites for protein folding, while this property of residual structure is replaced by the calcium-binding site between the domains in a-lactalbumin.

F 420 H 2 :NADP oxidoreductase is found in methanogenic, sulfate-reducing and halophilic archaea and also in some bacteria. The putative gene encoding the enzyme was cloned from Methanobacterium thermoautotrophicum (strain Marburg) and heterologously expressed in Escherichia coli. The overproduced active enzyme was purified, characterized and crystallized.z 1998 Federation of European Biochemical Societies.