Nucleotide sequence encoding the flavoprotein and hydrophobic subunits of the succinate dehydrogenase of Escherichia coli

Abstract

The nucleotide sequence of a 3614 base-pair segment of DNA containing the sdhA gene, encoding the flavoprotein subunit of succinate dehydrogenase of Escherichia coli, and two genes sdhC and sdhD, encoding small hydrophobic subunits, has been determined. Together with the iron-sulphur protein gene (sdhB) these genes form an operon (sdhCDAB) situated between the citrate synthase gene (gltA) and the 2-oxoglutarate dehydrogenase complex genes (sucAB): gltA-sdhCDAB-sucAB. Transcription of the gltA and sdhCDAB gene appears to diverge from a single intergenic region that contains two pairs of potential promoter sequences and two putative CRP (cyclic AMP receptor protein)-binding sites. The sdhA structural gene comprises 1761 base-pairs (587 codons, excluding the initiation codon, AUG) and it encodes a polypeptide of Mr 64268 that is strikingly homologous with the flavoprotein subunit of fumarate reductase (frdA gene product). The FAD-binding region, including the histidine residue at the FAD-attachment site, has been identified by its homology with other flavoproteins and with the flavopeptide of the bovine heart mitochondrial succinate dehydrogenase. Potential active-site cysteine and histidine residues have also been indicated by the comparisons. The sdhC (384 base-pairs) and sdhD (342 base-pairs) structural genes encode two strongly hydrophobic proteins of Mr 14167 and 12792 respectively. These proteins resemble in size and composition, but not sequence, the membrane anchor proteins of fumarate reductase (the frdC and frdD gene products).

Structural studies on three flavin‐interacting regions of the flavoprotein subunit of complex II in Ascaris suum mitochondria

“…1B) together with a fluorescent material characteristic of a flavin (emission at 520 nm when excited at 360 nm), indicating that the flavin in this subunit is covalently attached to the polypeptide, as is the case for bovine heart and bacterial Fp subunits [18,19]. The amino acid composition of the Asearis Fp subunit was similar to that of the bovine heart Fp subunit [20] and those deduced from nucleotide sequences of Fp subunits of E. coli sdhA [8] and frdA [10] (Table I). This similarity is reflected by the finding that antibodies raised against the Ascaris Fp subunit cross-reacted with the subunit of bovine heart complex II and that of E. co# complex II (sdhA) (data not shown).…”

“…The amino acid sequence around the histidyl residue is highly conserved between the bovine heart Fp subunit on the one hand and those of bacterial sdhA [8] andfrdA [10,13] on the other. To see if the amino acid sequence of the flavinbinding site of the Ascaris Fp subunit is also conserved, this subunit was digested with trypsin and the flavincontaining peptide was isolated and sequenced.…”

“…From a comparison of the primary structures of E. coli frdA and glutathione reductase, which also contains FAD as a prosthetic group, and of threedimensional structural data for them [8,10,22], it has been predicted that the AMP moiety of FAD in bacterial Fp subunits interacts noncovalently with two regions of the Fp polypeptide. One such region is called the flA-o~A-flB fold (Rossman nucleotide binding fold) [24], which is located near the NHz-terminus (e.g.…”

“…Another segment interacting with the AMP moiety is the stretch from residues 357 to 386 in E. coli sdhA [8] and from residues 355 to 374 in B. subtilis sdhA [11]. To see if this segment is also detectable in the Ascaris Fp subunit, 20 out of the 40 tryptic peptides of the Fp subunit were partially sequenced, since the Lys residues in the amino terminal part of the segment in bacterial Fp (Lys-343 in E. coli sdhA and Lys-341 in B. subtilis sdhA) are well-conserved and trypsin specifically digests the C-terminal of Lys and Arg residues in the a From the results reported in [20] b From the results reported in [8] c From the results reported in [10] a Calculated according to [21] peptide. It was thus possible to find a peptide showing high sequence similarity to the aforementioned segments of bacterial Fp subunits (Fig.…”

“…It is a major component of Ascaris mitochondria (8o70 of mitochondrial protein) [7] and is composed of the following four subunits: a flavoprotein (Fp) subunit containing a flavin (Mr= 68 kDa), an Ip subunit associated with iron-sulfur centers (Mr = 26 kDa) and two hydrophobic, heme bcontaining polypeptides called CybL and Cybs (Mr = 15 and 13.5 kDa, respectively) [3,5]. In the case of bacteria, the genes of complex II have been cloned and sequenced from Escherichia coil (sdh and frd) [8][9][10], Bacillus subtilis (sdh ) [11,12] and Proteus vulgaris (frd) [13]. Structural information on mitochondrial complex…”