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 00:00:00 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), Vol. 29:31; ISSN 0006-2960
• Country of Publication: United States
• Language: English

Related Subjects

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