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
Abstract
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 othermore »
- Authors:
-
- Harvard Medical School, Boston, MA (USA) Massachusetts Institute of Technology, Cambridge (USA)
- Ohio State Univ., Columbus (USA)
- Massachusetts Institute of Technology, Cambridge (USA)
- Harvard Medical School, Cambridge, MA (USA)
- Publication Date:
- OSTI Identifier:
- 6098337
- DOE Contract Number:
- FG02-87ER13731
- Resource Type:
- Journal Article
- Journal Name:
- Biochemistry; (USA)
- Additional Journal Information:
- Journal Volume: 29:31; Journal ID: ISSN 0006-2960
- Country of Publication:
- United States
- Language:
- English
- Subject:
- 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
Citation Formats
Alex, L A, Reeve, J N, Orme-Johnson, W H, and Walsh, C T. 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. United States: N. p., 1990.
Web. doi:10.1021/bi00483a011.
Alex, L A, Reeve, J N, Orme-Johnson, W H, & Walsh, C T. 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. United States. https://doi.org/10.1021/bi00483a011
Alex, L A, Reeve, J N, Orme-Johnson, W H, and Walsh, C T. 1990.
"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". United States. https://doi.org/10.1021/bi00483a011.
@article{osti_6098337,
title = {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},
author = {Alex, L A and Reeve, J N and Orme-Johnson, W H and Walsh, C T},
abstractNote = {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.},
doi = {10.1021/bi00483a011},
url = {https://www.osti.gov/biblio/6098337},
journal = {Biochemistry; (USA)},
issn = {0006-2960},
number = ,
volume = 29:31,
place = {United States},
year = {Tue Aug 07 00:00:00 EDT 1990},
month = {Tue Aug 07 00:00:00 EDT 1990}
}