A F420-dependent NADP reductase in the extremely thermophilic sulfate-reducing Archaeoglobus fulgidus |
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Authors: | Jasper Kunow Beatrix Schwörer Karl O Stetter Rudolf K Thauer |
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Institution: | (1) Laboratorium für Mikrobiologie des Fachbereichs Biologie der Philipps-Universität Marburg, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany;(2) Max-Planck-Institut für Terrestrische Mikrobiologie Marburg, Karl-von-Frisch-Strasse, D-35043 Marburg, Germany;(3) Lehrstuhl für Mikrobiologie, Universität Regensburg, Universitätsstrasse 31, D-93053 Regensburg, Germany |
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Abstract: | Archaeoglobus fulgidus, a sulfate-reducing Archaeon with a growth temperature optimum of 83°C, uses the 5-deazaflavin coenzyme F420 rather than pyridine nucleotides in catabolic redox processes. The organism does, however, require reduced pyridine nuclcotides for biosynthetic purposes. We describe here that the Archaeon contains a coenzyme F420-dependent NADP reductase which links anabolism to catabolism. The highly thermostable enzyme was purfied 3600-fold by affinity chromatography to apparent homogeneity in a 60% yield. The native enzyme with an apparent molecular mass of 55 kDa was composed of only one type of subunit of apparent molecular mass of 28 kDa. Spectroscopic analysis of the enzyme did not reveal the presence of any chromophoric prosthetic group. The purified enzyme catalyzed the reversible reduction of NADP (apparent K
M 40 M) with reduced F420 (apparent K
M 20 M) with a specific activity of 660 U/mg (apparent V
max) at pH 8.0 (pH optimum) and 80°C (temperature optimum). It was specific for both coenzyme F420 and NADP. Sterochemical investigations showed that the F420-dependent NADP reductase was Si face specific with respect to C5 of F420 and Si face specific with respect to C4 of NADP.Abbreviations F420
coenzyme F420
- F420H2
1,5-dihydrocoenzyme F420
- H4MPT
tetrahydromethanopterin
- CH=H4MPT
N5, N10-methylenetetrahydromethanopterin
- MFR
methanofuran
- HPLC
high performance liquid chromatography
- methylene-H4MPT dehydrogenase
N5, N10-methylenetetrahydromethanopterin dehydrogenase
- 1 U =
1 mol/min |
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Keywords: | Archaea Sulfate reducers Hyperthermophiles Coenzyme F420 Deazaflavin F420-dependent NADP reducatase Stereoselectivity Stereospecificity Archaeoglobus fulgidus |
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