The dinuclear paramagnetic center of the CuA variant of the cupredoxin amicyanin has been investigated using 1H NMR. The hyperfine-shifted resonances have been assigned using a combination of 1D NOE difference and 2D WEFT-NOESY spectroscopy. The shifts experienced by the assigned resonances have been used to calculate hyperfine coupling constants for these protons from which the spin density distribution on the ligands at the CuA center is obtained. A comparison with published data for the paramagnetic form of wild type amicyanin highlights a number of similarities and differences between these evolutionary related sites. In both cases 50-60% of the unpaired spin density is distributed on the ligands, which in the case of the CuA center involves two cysteine and two histidine ligands. The two weak axial interactions at the CuA center carry less than 1% spin density.
The kinetics and specificity of reductive acylation of lipoyl domains derived from Azotobacter vinelandii 2-oxo-acid dehydrogenase complexes, catalysed by A. vinelandii and Escherichia coli complexes, have been investigated. With the wild-type pyruvate dehydrogenase complex from A. vinelandii the rate of reductive acetylation and deacetylation was studied by rapid mixing methods. The rate of reductive acetylation, 126 s Ϫ1 , corresponds well with the turnover rate derived from steady-state measurements. Deacetylation was rapid and specific for coenzyme A. No deacetylation was observed with reduced or oxidised lipoamide or with dithiothreitol. The rate of reductive acetylation of complex-bound lipoyl domains by pyruvate dehydrogenase (E1p) is at least 60 times higher than of free lipoyl domains under comparable conditions. This gain in catalytic rate indicates a large diffusion limitation of lipoyl domains when attached via the flexible linker segments to the complex, and illustrates the efficiency of substrate channeling in the multienzyme complex.The 2-oxo-acid dehydrogenases exhibit specificity for lipoyl domains in the reductive acylation reaction. The A. vinelandii lipoyl domain derived from the pyruvate dehydrogenase complex is a good substrate for A. vinelandii E1p, but not for A. vinelandii 2-oxoglutarate dehydrogenase (E1o), and vice versa. The A. vinelandii lipoyl domain of the pyruvate dehydrogenase complex is also, although at a lower rate, reductively acetylated by E. coli E1p and reductively succinylated by E. coli E1o. Likewise, the A. vinelandii lipoyl domain derived from the 2-oxoglutarate dehydrogenase complex is recognised by E. coli E1o, but not by E. coli E1p. This suggests that common determinants of the lipoyl domains exist that are responsible for recognition by the E1 components. On the basis of the observed specificity and lipoyl domain sequences and structures, an exposed loop of the A. vinelandii 2-oxoglutarate dehydrogenase complex lipoyl domain was subjected to mutagenesis. Although the reductive acylation experiments of mutants of the lipoyl domain indicate the importance of this loop for recognition, it is probably not the single determinant for specificity.Keywords : multienzyme complex; lipoyl domain; recognition; mutagenesis; Azotobacter vinelandii.The 2-oxo-acid dehydrogenase multienzyme complexes ca-nase complex (OGDHC) converts 2-oxoglutarate into succinylCoA. The complexes effectuate these reactions by combining talyse the oxidative decarboxylation of 2-oxo acids to the correthe activities of three enzymes : a substrate-specific 2-oxo-acid sponding acyl-CoA derivatives, accompanied with the reduction dehydrogenase [pyruvate dehydrogenase (E1p) or 2-oxoglutarate of NAD ϩ [for recent reviews see Mattevi et al. (1992a), Berg dehydrogenase (E1o)], an acyltransferase [acetyltransferase and de Kok (1997b)]. In the gram-negative bacteria Azotobacter (E2p) or succinyltransferase (E2o)], and a common lipoamide vinelandii and Escherichia coli two similar complexes are predehydrogenase...
The three-dimensional structure of the N-terminal lipoyl domain of the acetyltransferase component of the pyruvate dehydrogenase complex from Azotobacter vinelandii has been determined using heteronuclear multidimensional NMR spectroscopy and dynamical simulated annealing. The structure is compared with the solution structure of the lipoyl domain of the A. vinelandii 2-oxoglutarate dehydrogenase complex. The overall fold of the two structures, described as a P-barrel-sandwich hybrid, is very similar. This agrees well with the high similarity of NMR-derived parameters, e.g. chemical shifts, between the two lipoyl domains. The main structural differences between the two lipoyl domains occur in a solventexposed loop close in space to the lipoylation site. Despite their high structural similarity, these lipoyl domains show a high preference for being reductively acylated by their parent 2-0x0 acid dehydrogenase. Potential residues of the lipoyl domain involved in this process of molecular recognition are discussed.Keywords: protein structure ; NMR; pyruvate dehydrogenase complex ; acetyltransferase ; molecular recognition.The pyruvate dehydrogenase complex (PDHC) catalyses the irreversible overall conversion of pyruvate to acetyl-CoA (for a recent review see Mattevi et al., 1992a). This multienzyme complex plays an important regulating role in the aerobic catabolism of carbohydrates, where it links glycolysis with the citric acid cycle. Pyruvate dehydrogenase complexes from Gram-negative bacteria are composed of multiple copies of three enzymic components ; pyruvate dehydrogenase (Elp), dihydrolipoyl acetyltransferase (E2p) and lipoamide dehydrogenase (E3). The structural core of these complexes consists of a cubic assemblage of 24 E2p chains arranged with octahedral symmetry. The E l p and E3 components are tightly but non-covalently bound to the E2p core as dimers. The 2-oxoglutarate dehydrogenase complex (OGDHC), which occurs in the citric acid cycle, belongs to the same family of 2-0x0 acid dehydrogenase complexes as the PDHC, as do the branched-chain 2-ox0 acid dehydrogenase complexes. These complexes all have similar structural and catalytic properties. The chain of the acetyltransferase component (E2p) of PDHC from A. vinelandii is, like other E2 components, highly segmented. It consists of five independently folded domains that are separated by long stretches (25-40 amino acids) of polypeptide chain which are flexible and unusually rich in alanine and proline residues (Hanemaaijer et al., 1988). From the N-terminus, the E2p chain consists of three highly similar lipoyl domains (about 80 residues) that each contain one covalently bound lipoic acid prosthetic group, a peripheral subunit-binding domain (about 35 residues), and a catalytic domain (29 kDa) which accommodates the acetyltransferase active site and is responsible for the formation of the core of the complex (Hanemaaijer et al., 1987).The lipoyl domains attached to the flexible linkers fulfil an indispensable role in coupling the three separate enzym...
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