Insights into the mechanism of ligand binding to octopine dehydrogenase from Pecten maximus by NMR and crystallography.

Octopine dehydrogenase (OcDH) from the adductor muscle of the great scallop, Pecten maximus, catalyzes the NADH dependent, reductive condensation of L-arginine and pyruvate to octopine, NAD(+), and water during escape swimming and/or subsequent recovery. The structure of OcDH was recently solved and a reaction mechanism was proposed which implied an ordered binding of NADH, L-arginine and finally pyruvate. Here, the order of substrate binding as well as the underlying conformational changes were investigated by NMR confirming the model derived from the crystal structures. Furthermore, the crystal structure of the OcDH/NADH/agmatine complex was determined which suggests a key role of the side chain of L-arginine in protein cataylsis. Thus, the order of substrate binding to OcDH as well as the molecular signals involved in octopine formation can now be described in molecular detail.

A structural basis for substrate selectivity and stereoselectivity in octopine dehydrogenase from Pecten maximus.

Octopine dehydrogenase [N(2)-(D-1-carboxyethyl)-L-arginine:NAD(+) oxidoreductase] (OcDH) from the adductor muscle of the great scallop Pecten maximus catalyzes the reductive condensation of l-arginine and pyruvate to octopine during escape swimming. This enzyme, which is a prototype of opine dehydrogenases (OpDHs), oxidizes glycolytically born NADH to NAD(+), thus sustaining anaerobic ATP provision during short periods of strenuous muscular activity. In contrast to some other OpDHs, OcDH uses only l-arginine as the amino acid substrate. Here, we report the crystal structures of OcDH in complex with NADH and the binary complexes NADH/l-arginine and NADH/pyruvate, providing detailed information about the principles of substrate recognition, ligand binding and the reaction mechanism. OcDH binds its substrates through a combination of electrostatic forces and size selection, which guarantees that OcDH catalysis proceeds with substrate selectivity and stereoselectivity, giving rise to a second chiral center and exploiting a "molecular ruler" mechanism.

Putative reaction mechanism of heterologously expressed octopine dehydrogenase from the great scallop, Pecten maximus (L).

cDNA for octopine dehydrogenase (ODH) from the adductor muscle of the great scallop, Pecten maximus, was cloned using 5'- and 3'-RACE. The cDNA comprises an ORF of 1197 nucleotides and the deduced amino acid sequence encodes a protein of 399 amino acids. ODH was heterologously expressed in Escherichia coli with a C-terminal penta His-tag. ODH-5His was purified to homogeneity using metal-chelate affinity chromatography and Sephadex G-100 gel filtration. Recombinant ODH had kinetic properties similar to those of wild-type ODH isolated from the scallop's adductor muscle. Site-directed mutagenesis was used to elucidate the involvement of several amino acid residues for the reaction catalyzed by ODH. Cys148, which is conserved in all opine dehydrogenases known to date, was converted to serine or alanine, showing that this residue is not intrinsically important for catalysis. His212, Arg324 and Asp329, which are also conserved in all known opine dehydrogenase sequences, were subjected to site-directed mutagenesis. Modification of these residues revealed their importance for the catalytic activity of the enzyme. Conversion of each of these residues to alanine resulted in strong increases in K(m) and decreases in k(cat) values for pyruvate and L-arginine, but had little effect on the K(m) and k(cat) values for NADH. Assuming a similar structure for ODH compared with the only available structure of a bacterial opine dehydrogenase, these three amino acids may function as a catalytic triad in ODH similar to that found in lactate dehydrogenase or malate dehydrogenase. The carboxyl group of pyruvate is then stabilized by Arg324. In addition to orienting the substrate, His212 will act as an acid-base catalyst by donating a proton to the carbonyl group of pyruvate. The acidity of this histidine is further increased by the proximity of Asp329.

Organophosphate-resistant forms of acetylcholinesterases in two scallops--the Antarctic Adamussium colbecki and the Mediterranean Pecten jacobaeus.

We describe the acetylcholinesterase polymorphisms of two bivalve molluscs, Adamussium colbecki and Pecten jacobaeus. The research was aimed to point out differences in the expression of pesticide-resistant acetylcholinesterase forms in organisms living in different ecosystems such as the Ross Sea (Antarctica) and the Mediterranean Sea. In A. colbecki, distinct acetylcholinesterase molecular forms were purified and characterized from spontaneously soluble, low-salt-soluble and low-salt-Triton extracts from adductor muscle and gills. They consist of two non-amphiphilic acetylcholinesterases (G(2), G(4)) and an amphiphilic-phosphatidylinositol-membrane-anchored form (G(2)); a further amphiphilic-low-salt-soluble G(2) acetylcholinesterase was found only in adductor muscle. In the corresponding tissues of P. jacobaeus, we found a non-amphiphilic G(4) and an amphiphilic G(2) acetylcholinesterase; amphiphilic-low-salt-soluble acetylcholinesterases (G(2)) are completely lacking. Such results are related with differences in cell membrane lipid compositions. In both scallops, all non-amphiphilic AChEs are resistant to used pesticides. Differently, the adductor muscle amphiphilic forms are resistant to carbamate eserine and organophosphate diisopropylfluorophosphate, but sensitive to organophoshate azamethiphos. In the gills of P. jacobaeus, amphiphilic G(2) forms are sensitive to all three pesticides, while the corresponding forms of A. colbecki are sensitive to eserine and diisopropylfluorophosphate, but resistant to azamethiphos. Results indicate that organophosphate and/or carbamate resistant AChE forms are present in species living in far different and far away environments. The possibility that these AChE forms could have ensued from a common origin and have been spread globally by migration is discussed.