PMID:8075801

Citation	Williams, R, Cotton, NP, Thomas, CM and Jackson, JB (1994) Cloning and sequencing of the genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum and the implications for the domain structure of the enzyme. Microbiology (Reading, Engl.) 140 ( Pt 7):1595-604
Abstract	The genes for the proton-translocating nicotinamide nucleotide transhydrogenase from Rhodospirillum rubrum have been cloned using a probe constructed with the polymerase chain reaction, genomic DNA as target and oligonucleotide primers corresponding to amino acid sequence obtained from the purified soluble subunit. There is a cluster of three genes, designated pntAA, pntAB and pntB, whose translation products indicate polypeptides of 384, 139 and 464 amino acids, respectively. This contrasts with the situation in the enzymes from Escherichia coli (two polypeptides) and bovine mitochondria (one polypeptide) but there is close similarity between the sequences. PntAA is the soluble subunit of the enzyme from R. rubrum, equivalent to the relatively hydrophilic domain I that forms the N-terminal part of the alpha polypeptide of E. coli transhydrogenase and which probably contains the NAD(H)-binding site. PntAB corresponds to the strongly hydrophobic domain IIa at the C-terminus of the alpha polypeptide of the E. coli transhydrogenase. PntB corresponds to the E. coli beta polypeptide, which comprises the strongly hydrophobic domain IIb and the relatively hydrophilic domain III, thought to contain the NADP(H)-binding site. The peptide bond between PntAA-Lys237 and -Glu238 of both the denatured and the native soluble subunit is very sensitive to proteolysis by trypsin and the neighbouring peptide bond Lys227-Thr228 to cleavage by the endoproteinase Lys-C. Related sites have been reported to be sensitive to trypsin in the E. coli and bovine mitochondrial enzymes. The two tryptic fragments from the native R. rubrum soluble subunit are unable to reconstitute transhydrogenase activity to membranes depleted of the soluble subunit but they can block reconstitution by intact soluble subunit. It is suggested that this protease-sensitive region separates two subdomains and that, after trypsinolysis, at least one retains structural integrity and can dock with domains II and/or III.
Links	PubMed
Keywords	Amino Acid Sequence; Base Sequence; Binding Sites; Cloning, Molecular; Genes, Bacterial/genetics; Light; Molecular Sequence Data; NAD/metabolism; NADP/metabolism; NADP Transhydrogenases/genetics; NADP Transhydrogenases/metabolism; NADP Transhydrogenases/radiation effects; Peptide Fragments/metabolism; Protein Conformation; Protons; Rhodospirillum rubrum/enzymology; Rhodospirillum rubrum/genetics; Sequence Analysis, DNA; Sequence Homology, Amino Acid
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Annotations

Gene product	Qualifier	GO Term	Evidence Code	with/from	Aspect	Extension	Notes	Status
RHORT:PNTAA		GO:0008750: NAD(P)+ transhydrogenase (AB-specific) activity	ECO:0000250:	PMID:8232423	F		Figure 3: Comparison of the domain and polypeptide structures of H+-Thase from R.rubrum, E.coli, bovine mitochondria and Ei.tenella and with alanine dehydrogenase from Bacillus stearothermophilus.	complete CACAO 3299
RHORT:PNTAA		GO:0008750: NAD(P)+ transhydrogenase (AB-specific) activity	ECO:0000314:		F		Figure 6.	complete CACAO 3451
RHORT:PNTAA	enables	GO:0008750: NAD(P)+ transhydrogenase (AB-specific) activity	ECO:0000314: direct assay evidence used in manual assertion		F		Seeded From UniProt	complete

References

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