Biotinylation Domain Research Articles

Cloning, sequencing and expression of rat liver pyruvate carboxylase

Overlapping clones encoding rat liver pyruvate carboxylase (PC) have been isolated by screening a liver cDNA library and by performing rapid amplification of cDNA ends polymerase chain reaction on total liver RNA. The sequence of rat PC cDNA contains an open reading frame of 3537 nucleotides encoding a polypeptide of 1178 amino acids with a calculated M(r) of 129848. This is flanked by a 5' untranslated region of 66 bp and a 3' untranslated region of 421 bp including the poly(A) tail. The inferred protein sequence is 96.6% identical with mouse and 96.3% identical with human PCs, 68.4% identical with mosquito PC and 53.5% identical with yeast PC isoenzymes PC1 and PC2. On the basis of partial proteolysis and sequence homology with PC from other organisms (yeast, mosquito, mouse and human) and with other biotin enzymes, three functional domains, namely the biotin carboxylation domain, the transcarboxylation domain and the biotinyl domain, have been identified. Comparison with the known structure of the biotin carboxylase subunit of Escherichia coli acetyl-CoA carboxylase [Waldrop, Rayment and Holden (1994) Biochemistry 33, 10249-10256] highlights the functional importance of 11 highly conserved residues. Northern analysis revealed that PC mRNA is highly expressed in rat liver, kidney, adipose tissue and brain, moderately expressed in heart, adrenal gland and lactating mammary gland, and expressed at a low level in spleen and skeletal muscle.

The major biotinyl protein from Pisum sativum seeds covalently binds biotin at a novel site.

Seeds of Pisum sativum contain a biotinyl polypeptide called SBP65 that behaves as a putative sink for the free vitamin, representing more than 90% of the total protein-bound biotin in mature seeds. A cDNA encoding SBP65 was cloned and sequenced. The deduced primary structure of the protein was confirmed by protein sequencing. Peptide sequencing also indicated binding of the biotin to lysine 103. The biotinylation domain of SBP65 differs markedly from that of presently known biotin enzymes. Molecular analysis of the protein sequence reveals an extremely hydrophilic protein containing several repeated motifs. These properties, as well as the temporal and spatial patterns of expression of this protein, suggest that SBP65 belongs to the LEA (late embryogenesis-abundant) group of proteins.

Structural Dependence of Post-translational Modification and Reductive Acetylation of the Lipoyl Domain of the Pyruvate Dehydrogenase Multienzyme Complex

The lipoyl domain of the dihydrolipoyl acetyltransferase (E2) component of the pyruvate dehydrogenase multienzyme complex is recognized specifically by the lipoylating enzyme(s) in the cell and by the pyruvate dehydrogenase (E1) component in the parent complex. Highly conserved aspartic acid and alanine residues flank the lipoyl-lysine residue, on the N and C-terminal sides, respectively, in the sharp β-turn in which the lipoyl-lysine residue is prominently displayed. A sub-gene encoding the lipoyl domain of the Bacillus stearothermophilus pyruvate dehydrogenase complex was subjected to mutagenesis in the vector M13mp18. Aspartic acid 41 was changed to glutamic acid (D41E), alanine (D41A) and lysine (D41K), and alanine 43 was changed to methionine (A43M), lysine (A43K) and glutamic acid (A43E). The double mutations D41KK42A and D41MA43M were also made. All mutant domains were capable of being lipoylated, apart from the D41KK42A domain where the lipoyl-lysine had been moved round the β-turn by one position towards the N terminus. Neither the D41K nor the A43K mutants showed any doubly lipoylated domain and the single lipoyl group was found attached only to the correct lysine residue. Accurate positioning of the lipoyl-lysine in the β-turn is thus are essential cue for lipoylation, but the conserved aspartic acid and alanine residues are not necessary for the domain to be recognized by the lipoylating enzyme(s). No biotinylation of the D41MA43M mutant domain was observed, although the sequence motif MKM is highly conserved as the biotinylation site in the structurally homologous biotinyl domain of biotin-containing enzymes. The mutations at the aspartic acid 41 position all lowered the rate of reductive acetylation of the lipoyl domain by the E1 component of the pyruvate dehydrogenase complex, as did the mutations A43E and A43K. The A43M mutant was reductively acetylated at the same rate as the wild-type domain. Thus, both the alanine and aspartic acid residues are important for recognition of the domain by E1, but there is no absolute dependence on retention of the sequence surrounding the lipoyl-lysine residue.

Chimeric proteins incorporating an In vivo biotinylation domain

Chimeric proteins incorporating an In vivo biotinylation domain

Interaction of avidin with the lipoyl domains in the pyruvate dehydrogenase multienzyme complex: three-dimensional location and similarity to biotinyl domains in carboxylases.

Avidin can form intermolecular cross-links between particles of the pyruvate dehydrogenase multienzyme complex from various sources. Avidin does this by binding to lipoic acid-containing regions of the dihydrolipoamide acetyltransferase polypeptide chains that comprise the structural core of the complex. It is inferred that the lipoyl domains of the acetyltransferase chain extend outwards from the interior of the enzyme particle, interdigitating between the subunits of the other two enzymes bound peripherally in the assembled structure, with the lipoyl-lysine residues capable of reaching to within at least 1-2 nm of the outer surface of the enzyme complex (diameter ca. 37 nm). The distribution of enzymic activities between different domains of the dihydrolipoamide acetyltransferase chain implies that considerable movement of the lipoyl domains is a feature of the catalytic activity of the enzyme complex. There is evidence that the lipoyl domain of the 2-oxo acid dehydrogenase complexes is similar in structure to a domain that binds the cofactor biotin, also in amide linkage with a specific lysine residue, in the biotin-dependent class of carboxylases.