Sequence conservation in the α and β subunits of pyruvate dehydrogenase and its similarity to branched‐chain α‐keto acid dehydrogenase

Abstract

Amintr acid sequence ecmp~risan of'8 tl nnd 6 JsubunEta of the a-kcta aeici dchydrngenanc (E,] component af thr pyruvutc dehydruacnuac complcr and brenehcd-ehrrin wkc~n ticid dckydroganurc enmglox from multiple xpcies was performed by cemputcr analysis. In addition to 2 prcviaullly reeegnircd rcpianr ot'hamaisgy in the 01 subunit, a 3rd rct$an oP cxtcnrlvc homolapy was identified in E,a, and miry bc one of tht? riru invDlvcd in subunit interaction. S,fl containr 4 rcyionr of extensive homology. Region I contains IO amino nridr that WC Ramolo#juur; ta a I&amino acid stretch in Ekh~rieiritr rdi/i E,. Regianr 1 and 3 hrvc scquencc homoiagicn with ether dehydregcnam~ sugyrrting that rhcro fcgionr may be involved in eatnlywix.Byruvntc dchydrogcnaxc: Pyruvntc dchydrelgenttbc complex: Urrnehcd-chain a&eta acid dchydrogcnusc complex: cl-Keto acid dchydregcnrsc corns plcx; Amino acid sequcncccomparioen INTRODUCTIONThe mammalian pyruvate dehydrogcnase complex (PDC) is a mitochondrial multienzyme complex that ' catalyzes the conversion of pyruvate to acetyl-CoA [1,2]. The complex consists of 3 catalytic and 2 regulatory components, and a protein X component. In plant {3], eukaryotic, and some prokaryotic species [4], the first catalytic component, pyruvate dehydrogcnase, (El) (py, uvate: lipoamide 2.oxidoreductase, EC 1.2.4.1), is composed of 2 non-identical 01 and P subunits which form a heterotetramer. El, which requires thiamin pyrophosphate as a cofactor, catalyzes the oxidative decarboxylation of pyruvate according to the following 2 partial reactions.amino acid residues Elas identified critical amino acids in the El heterotetramer that are critical For enzyme function [7]. Recent studies have shown that El& but not ElcrI is protected from trypsin digestion when bound to dihydrolipoyl acetyltransferase (I%), the 2nd catalytic component of PDC, suggesting that EIP binds to EZ [8]. Structure-function analysis has been facilitated by the cloning of both the (Y [4,9-111 and I3 [4,12,13] subunits of PDC from multiple species. However, the critical amino acid residues in the functional domains of E,DL and I&P remain unidentified. In the present study, we have used computer analysis to identify several regions of the (Y and /3 subunits that are highly conserved not only in all the known sequences of the 2 subunits of PDC from various species, but also in those of the branched-chain ol-keto acid dlhydrogenase complexes (BCKDC) in both prokaryotic and eukaryotic species. Similarities with other proteins suggest that one of the conserved domains may play a critical role in the oxidative decarboxylation of the 01-keto acids. Based on limited data, it has been postulated that reaction (I) is carried out by the (v subunit, and reaction (ii) by the P subunit [5]. Etu! activity is regulated by phosphorylation-dephosphorylation by a specific El kinase and phosphatase [l]. Sequence comparison of multiple thiamine-dependent enzymes has allowed for the identification of a putative thiamin gyrophosphate binding motif in Ela, [6]. Chemica...

Impaired Assembly of E1 Decarboxylase of the Branched-chain α-Ketoacid Dehydrogenase Complex in Type IA Maple Syrup Urine Disease

“…This unstable conformation is also manifested by the apparent dissociation of the mutant tetramers to lower molecular weight species as detected by sucrose density gradient centrifugation. The current data indicate that the T265R-␣ residue also plays a key role in subunit interactions and are consistent with the location of this residue at the putative subunit-interaction site conserved between BCKD and pyruvate dehydrogenase E1 proteins (29).…”

Cloning and sequencing of a cluster of genes encoding branched-chain alpha-keto acid dehydrogenase from Streptomyces avermitilis and the production of a functional E1 [alpha beta] component in Escherichia coli

“…A computer homology search of translated nucleotide and peptide sequence databases by using the deduced amino acid sequence of the S. avermitilis ORF1 and ORF2 sequences showed the best scores with E1␣ and E1␤ subunits of several BCDH and PDH complexes from different species (Table 1). A multiple amino acid sequence alignment of the S. avermitilis ORF1 product with those of the E1␣ BCDH and E1␣ PDH subunits in Table 1 showed similarity to structural motifs found in all E1␣ subunits of ␣-keto acid dehydrogenase complexes examined so far, such as the TPPbinding motif (25), a putative subunit interaction site (65), and the phosphorylation sites (I and II) of the E1␣ chains of the mammalian BCDH and PDH complexes (12,47,70) (Fig. 2).…”

“…2). Similarly, alignment of the ORF2 product with the E1␤ BCDH and E1␤ PDH subunits revealed four regions of extensive similarity characteristic of this protein family (65) (Fig. 2).…”

“…The deduced amino acid sequence is given in the single-letter code below the nucleotide sequence. Three regions of homology in the ␣ subunit and four regions of homology in the ␤ subunit of the E1 component of the BCDH complex, as identified by Wexler et al (65), are indicated by dashed lines below the deduced amino acid sequences. Amino acid residues identical in the S. avermitilis E1␣ and E1␤ proteins and at least one other E1␣ or E1␤ protein (see Table 1 for references) are indicated in boldface.…”

“…To further understand the involvement of the BCDH activity in avermectin production, we decided to clone and analyze the genes encoding BCDH from S. avermitilis. Recently, an amino acid sequence comparison of all of the published sequences for both E1␣ and E1␤ subunits of the PDH and the BCDH complexes from multiple species was performed by computer analysis (65). Interestingly, several regions of the ␣ and ␤ subunits were identified that are highly conserved not only in all PDHs so far described but also in both prokaryotic and eukaryotic BCDH complexes.…”