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

Journal Article · Tue Aug 07 04:00:00 EDT 1990 · Biochemistry; (USA)

DOI:https://doi.org/10.1021/bi00483a011· OSTI ID:6098337

Alex, L A ^[1]; Reeve, J N ^[2]; Orme-Johnson, W H ^[3]; Walsh, C T ^[4]

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1. Harvard Medical School, Boston, MA (USA) Massachusetts Institute of Technology, Cambridge (USA)
2. Ohio State Univ., Columbus (USA)
3. Massachusetts Institute of Technology, Cambridge (USA)
4. Harvard Medical School, Cambridge, MA (USA)

The genes frhA (1,217 bp), frhB (845 bp), and frhG (710 bp) encoding the three known subunits, {alpha}, {beta}, and {gamma}, of the 8-hydroxy-5-deazaflavin (F{sub 420}) 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 H{sub 2} 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 they 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.

• DOE Contract Number: FG02-87ER13731
• OSTI ID: 6098337
• Journal Information: Biochemistry; (USA), Journal Name: Biochemistry; (USA) Vol. 29:31; ISSN 0006-2960; ISSN BICHA
• Country of Publication: United States
• Language: English

Related Subjects

550601* -- Medicine-- Unsealed Radionuclides in Diagnostics
62 RADIOLOGY AND NUCLEAR MEDICINE
ACRIDINES
AMINES
AROMATICS
AUTORADIOGRAPHY
AZAARENES
AZINES
BACTERIA
BETA DECAY RADIOISOTOPES
BETA-MINUS DECAY RADIOISOTOPES
CLONING
DAYS LIVING RADIOISOTOPES
DNA HYBRIDIZATION
DNA SEQUENCING
DNA-CLONING
ENZYMES
EVEN-ODD NUCLEI
FLAVINES
GENE REGULATION
GENES
HETEROCYCLIC COMPOUNDS
HYBRIDIZATION
HYDROGENASES
ISOTOPES
LIGHT NUCLEI
METHANOGENIC BACTERIA
MICROORGANISMS
NUCLEI
ORGANIC COMPOUNDS
ORGANIC NITROGEN COMPOUNDS
OXIDOREDUCTASES
PEPTIDES
PROTEINS
PYRIDINES
RADIOISOTOPES
STRUCTURAL CHEMICAL ANALYSIS
SULFUR 35
SULFUR ISOTOPES