Cloning, sequence determination, and expression of the genes encoding the subunits of the nickel-containing 8-hydroxy-5-deazaflavin reducing hydrogenase from Methanobacterium thermoautotrophicum delta H.
The genes frhA (1217 bp), frhB (845 bp), and frhG (710 bp) encoding the three known subunits, alpha, beta, and gamma, of the 8-hydroxy-5-deazaflavin (F420) reducing hydrogenase (FRH) from the thermophilic methanogen Methanobacterium thermoautotrophicum delta H have been cloned, sequenced, and shown to be tightly linked, indicative of a single transcriptional unit. The DNA sequence contains a fourth open reading frame, designated frhD (476 bp), encoding a polypeptide (delta) that does not copurify with the active enzyme. Expression of the frh gene cluster in Escherichia coli shows that four polypeptides are synthesized. When analyzed by SDS-PAGE, the proteins migrate with mobilities consistent with their calculated molecular weights. In order to understand the mechanism of H2 oxidation by this enzyme, localization of redox cofactors (Ni, Fe/S, FAD) to specific subunits and information on their structure is needed. This has been hindered due to the refractory nature of the enzyme to denaturation methods needed in order to obtain individual subunits with cofactors intact. In this paper we discuss the possible localization of the redox cofactors as implicated from the DNA-derived protein sequences of the subunits. The amino acid sequences of the subunits of the FRH are compared with those of other Ni-containing hydrogenases, including the methyl viologen reducing hydrogenase (MVH) of M. thermoautotrophicum delta H.
Alex
L A
LA
Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, Massachusetts 02115.
Reeve
J N
JN
Orme-Johnson
W H
WH
Walsh
C T
CT
D000595:Amino Acid Sequence; D001426:Bacterial Proteins / Q000096:biosynthesis / Q000235:genetics*; D001483:Base Sequence; D004790:Enzyme Induction; D019605:Euryarchaeota; D005798:Genes, Bacterial; D008969:Molecular Sequence Data; D009247:NADH, NADPH Oxidoreductases / Q000096:biosynthesis / Q000235:genetics*; D016366:Open Reading Frames; D011993:Recombinant Fusion Proteins / Q000235:genetics; D016415:Sequence Alignment; D012689:Sequence Homology, Nucleic Acid
D001426:Bacterial Proteins; D011993:Recombinant Fusion Proteins; D009247:NADH, NADPH Oxidoreductases; C058515:8-hydroxy-5-deazaflavin-NADPH oxidoreductase