Sequence analysis of the lpdV gene for lipoamide dehydrogenase of branched-chain-oxoacid dehydrogenase of Pseudomonas putida.
The production of two lipoamide dehydrogenases by Pseudomonas is so far unique. One, LPD-val, is the specific E3 component of the branched-chain-oxoacid dehydrogenase and the second, LPD-glc, is the E3 component of 2-oxoglutarate dehydrogenase and the L-factor of the glycine oxidation system. The objective of the present research was to determine the nucleotide sequence of the structural gene for LPD-val in order to compare its deduced amino acid structure with that of other redox-active disulfide flavoproteins. Northern blots using mRNA isolated from P. putida grown in media with branched-chain amino acids identified a transcript of 6.2 kb which is long enough to encode all the structural genes for the complex. The nucleotide sequence of the structural gene for LPD-val, lpdV, was determined and consists of 459 codons plus the stop codon. The open reading frame begins two bases after the stop codon for the E2 subunit and is composed of 66.3% G + C. Codon usage is characteristic of moderately strongly expressed genes. There is a ribosome-binding site preceding the ATG start codon and a strong candidate for a rho-independent terminator at the 3' end of the reading frame. The Mr of the protein encoded is 48,164 and when the Mr of FAD is added, the total Mr is 48,949, which is very close to the value of 49,000 obtained by SDS-polyacrylamide gel electrophoresis. Similarity comparisons of LPD-val with sequences of three other lipoamide dehydrogenases showed that LPD-val was somewhat more distantly related. It is probable that the lipoamide dehydrogenases and the glutathione and mercuric reductases evolved from a common ancestral flavoprotein.
Burns
G
G
Department of Biochemistry and Molecular Biology, University of Oklahoma Health Sciences Center, Oklahoma City 73190.
Brown
T
T
Hatter
K
K
Sokatch
J R
JR
D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D001483:Base Sequence; D015152:Blotting, Northern; D003062:Codon; D008058:Dihydrolipoamide Dehydrogenase / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D005796:Genes; D005798:Genes, Bacterial; D007658:Ketone Oxidoreductases / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000032:analysis / Q000096:biosynthesis / Q000235:genetics*; D010088:Oxidoreductases / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*; D012333:RNA, Messenger / Q000032:analysis
D000596:Amino Acids; D003062:Codon; D009097:Multienzyme Complexes; D012333:RNA, Messenger; D010088:Oxidoreductases; C021551:mercuric reductase; D007658:Ketone Oxidoreductases; D042942:3-Methyl-2-Oxobutanoate Dehydrogenase (Lipoamide); D008058:Dihydrolipoamide Dehydrogenase