Identification of octopine dehydrogenase from Mytilus galloprovincialis

A cDNA encoding the putative octopine dehydrogenase (OcDH) from the mussel Mytilus galloprovincialis was cloned and sequenced. The complete coding region was expressed in the bacteria Escherichia coli and the recombinant protein was purified. The M. galloprovincialis OcDH appears to have the highest affinity for the amino acid substrate L-arginine (88.22%), compared to L-alanine (9.04%) and glycine (2.74%). This enzyme showed no activity when taurine or β-alanine was used as substrate. These data strongly support that this recombinant enzyme is octopine dehydrogenase and not another opine dehydrogenase such as alanopine or strombine dehydrogenases. The superimposition of the theoretical three-dimensional model of the M. galloprovincialis OcDH and the crystal structure of its homologous counterpart from the great scallop Pecten maximus showed interesting changes in the amino acid binding site which could explain the differences found in the substrate affinity between the two molluscs. A phylogenetic analysis was performed comparing M. galloprovincialis OcDH and annotated sequences representing the five opine dehydrogenase (OpDH) protein family members. The phylogenetic tree which was obtained clustered the OpDH enzymes according to the evolutionary relationships of the species and not to the biochemical reaction catalysed. Octopine dehydrogenase has been identified in the Mytilidae family for the first time, having previously only been established in one other marine invertebrate (P. maximus).     

Temperature-determined enzymatic functions in octopine dehydrogenase.

We investigated the temperature dependence of several functions of octopine dehydrogenase, a monomeric enzyme extracted from the shell fish Pecten maximus L. We found that six enzymatic functions are temperature independent or change only negligibly with temperatue. These are the dissociation constants of three coenzyme complexes and the Michaelis Km values for NAD, NADH and one of the substrates (D-octopine). This is taken as an indication of a temperature-regulatory mechanism which enables the enzyme to maintain a constant level of NAD, NADH and D-octopine in binary and ternary complexes independent of fluctuations of the external temperature. This is discussed with reference to enzymes from other poikilotherms, which reportedly display similar biologically meaningful response to temperature. We also discuss the meaning of our data from a thermodynamic viewpoint. Considering that in a temperature-independent binding process only entropy changes contribute to the standard free-energy change, we speculate on possible molecular models which might account for our results. We also investigate the activation-energy parameters for the reaction catalyzed by octopine dehydrogenase, as obtained from the temperature dependence of V. It is found that octopine dehydrogenase, relative to other dehydrogenases, is provided with a rather low delta H not equal to, which enables the enzyme to change its turnover number by only a small factor in the temperature range 5--35 degrees C.     

Bromopyruvate, a potential affinity label for octopine dehydrogenase

Bromopyruvate, an analogue of pyruvate, one of the substrates of octopine dehydrogenase, was tested as an inhibitor of the enzyme. Provided both the coenzyme and the second substrate, arginine, were present, bromopyruvate rapidly inactivated the enzyme. This inactivation was irreversible, obeyed pseudo-first order kinetics and exhibited a rate saturation effect. Pyruvate protected the enzyme against inactivation by bromopyruvate and these compounds competed for the same site. Bromopyruvate also behaved as a true substrate for the enzyme. This reagent thus exhibits the kinetic characteristics of a good affinity label for octopine dehydrogenase.     

Isolation, physicochemical properties, and folding of octopine dehydrogenase from Pecten jacobaeus

Two types (isoenzymes) of octopine dehydrogenase (A and B) from Pecten jacobaeus adductor muscle were purified to homogeneity, applying affinity chromatography as an efficient final step of purification. Both forms of the enzyme differ in their electrophoretic mobility. All other physico-chemical and enzymatic properties, as well as the folding behaviour were found to be identical. Interconversion of one form into the other was not detectable. Sedimentation equilibrium, gel permeation chromatography, and NaDodSO4/polyacrylamide gel electrophoresis yield a relative molecular mass of 45000 +/- 1500 for both native and denatured enzyme. The unfolding transition at varying guanidine X HCl concentrations is characterized by a two-step profile: at 0.4-0.8 M, partial unfolding is parallelled by inactivation; at 2.0-2.4 M the residual structure is destroyed in a second unfolding step. Beyond 2.8 M no further changes in fluorescence emission and dichroic absorption are observed. At 0.4-1.8 M guanidine X HCl, partial unfolding is superimposed by aggregation. The emission maximum of the intrinsic protein fluorescence at 327 nm is shifted to 352 nm upon denaturation in 6 M guanidine X HCl. Changes in the far-ultraviolet circular dichroism indicate complete loss of the overall backbone structure in this denaturant, including the native helix content of about 33%. Denaturation in 6 M guanidine X HCl, as monitored by the decrease of protein fluorescence, is fast (less than 8s). Upon reactivation after short denaturation, about 25% of the activity is recovered in a fast initial phase (less than 20s). The product of this phase has a similar stability towards destabilizing additives or proteases as the native enzyme. The slow phase of reactivation, which predominates after long-term denaturation, is determined by a single first-order reaction characterized by tau = 29 +/- 3 min (20 degrees C). This reaction must be a relatively late event on the folding pathway, preceded by the fast formation of a structured intermediate, as indicated by the immediate recovery of the native fluorescence. The structural rearrangements, which are rate-limiting for reactivation after long-term denaturation, are characterized by a high energy of activation (112 +/- 8 kJ/mol). The slow reactivation step is compatible in rate with the first-order folding reactions involved in the reconstitution of several oligomeric dehydrogenases [c.f. R. Jaenicke and R. Rudolph (1983) Colloq. Ges. Biol. Chem. Mosbach 34, 62-90].     

Fluorescence properties of reduced thionicotinamide--adenine dinucleotide and of its complex with octopine dehydrogenase.

Reduced 3-thionicotinamide--adenine dinucleotide (sNADH) is shown to be fluorescent, with an emission maximum at 510 nm when excited in the region of the absorption maximum (398 nm), and with a very low quantum yield, (3.4 +/- 0.5) x 10(-4). The interaction between sNADH and octopine dehydrogenase was investigated by ultraviolet-difference spectroscopy and fluorescence. Some surprising fluorescence features were found when sNADH was bound to the enzyme in the presence of D-octopine, as follows. (a) There is an unusually high enhancement of the dinucleotide fluorescence (by at least a factor of 100) attended by a 40-nm blue shift of the emission maximum. (b) The protein fluorescence is quenched almost completely. (c) The bound coenzyme analog undergoes a photoreaction, which proceeds differently from that occurring the free form. These features appear to be unique to the octopine.sNADH complex, as for example they are not present when sNADH is bound to horse liver alcohol dehydrogenase, or when NADH is bound to octopine dehydrogenase. The possible origin of these fluorescence features is discussed. Binding and kinetic studies were carried out with sNAD and sNADH. It was found that sNAD neither binds nor acts kinetically as a coenzyme. sNADH exhibits relatively good binding, with Km and Ki values close to those of the natural coenzyme, but the turnover number is 460 times smaller than that with NADH. The kinetic consequences of these findings are discussed. The sNADH dissociation constants were determined as a function of temperature, and appear to be practically temperature-independent in the range 10--40 degrees C. It seems thus, in agreement with previous studies, that the interaction between octopine dehydrogenase and coenzymes proceeds athermically, regardless of the structure, affinity, and chemical reactivity of the coenzyme. The possible biological and chemical meaning of this finding is discussed.     

Investigation on the kinetic mechanism of octopine dehydrogenase. A regulatory behavior.

The kinetic scheme of octopine dehydrogenase of Pecten maximus L., a monomeric enzyme obeying a bi-ter sequential mechanism, was completed, essentially in the forward reaction, by steady-state studies over a wide range of substrate concentration at pH 7.0. Deviation from the Michaelis-Menten behavior with respect to NAD+ and other significant kinetic data led us to ascribe for octopine dehydrogenase mechanism the mnemonical enzyme concept. In addition, another regulatory behavior can be envisaged involving the formation of two dead-end complexes enzyme.NADH.D-octopine and enzyme.NAD+.pyruvate.L-arginine.     

Investigations on the kinetic mechanism of octopine dehydrogenase. 1. Steady-state kinetics.

The kinetic mechanism of action of octopine dehydrogenase was investigated. This enzyme catalyses the reversible dehydrogenation of D-octopine to L-arginine and pyruvate, in the presence of nicotinamide-adenine dinucleotide. Initial velocity and product inhibition studies were carried out in both directions. Most of the results are consistent with a bi-ter sequential mechanism where NAD+ binds first to the enzyme followed by D-octopine, and the products are released in the order L-arginine, pyruvate and NADH. Various kinetic parameters were determined for each reactant at 33 degrees C, at pH 9.6 for NAD reduction, at pH 6.6 for NADH oxidation.     

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

Octopine dehydrogenase [N²-(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.     

Polymorphisms of octopine dehydrogenase (Odh) in mollusks and implications for the neutralism-selectionism hypothesis

Octopine dehydrogenase (Odh) was examined in several species of bivalves and gastropods and complemented with bibliographic data, to assess the controversy between neutralism and selectionism in explaining the maintenance of genetic variation in natural populations. This debate was the center of the molecular evolution and population genetic research in the 1970s and 1980s, but waned thereafter, without resolution. Although DNA data have been produced, implications are not understood. We examined the polymorphims of Odh in several species of bivalves and gastropods, and the kinetic properties (apparent Km) of the different isozymes in the scallop Euvola ziczac that indicates an apparent case of overdominance of the heterozygous individuals. The question "which of the two hypothesis is correct" has shifted with time to "how much influence did each factor have in the maintenance of genetic variation".     

Purification and characterization of two allozymic forms of octopine dehydrogenase from California populations ofMetridium senile

Summary In northern and southern California populations of the plumose sea anemone,Metridium senile, octopine dehydrogenase occurs in two allozymic forms and these forms are distributed in a highly population-specific manner; the frequency of the slow allele (ODH ¹⁰⁰) is 0.875 in the northern (Bodega Bay) population while the frequency of the fast allele (ODH ¹⁰³) in the southern population (Santa Barbara) is 0.125. Purification techniques resulted in an increase in purity of approximately 400 fold. The enzyme is a monomer ofM _r 35,000 to 40,000. Though there is some flexibility in the amino acid substrate which the enzyme uses (arginine and lysine react similarly), the specificity for the keto acid is limited to pyruvate.The kinetic characters of the two allozymes ofMetridium senile ODH are very different with respect to type of substrate saturation (Fig. 4 and 5) and apparent Michaelis constants (K _m) for pyruvate, lysine, arginine, and octopine (Table 5), product inhibition by octopine (Fig. 2), and optimal activity with respect to pH (Fig. 3). The properties of the slow and fast allozymes resemble the kinetic properties of cephalopod brain and muscle tissue-specific isozymes (Table 7). The kinetic data indicate that the slow allozyme would not allow a great deal of accumulation of octopine in vivo, while the fast allozyme is poised markedly towards octopine production.When the data presented in this study are compared to various physiological findings of other investigators, it becomes evident that the probable in vivo function of ODH in sea anemones is to act, in a manner analogous to vertebrate LDH, during the short-term anaerobiosis associated with muscle contraction and locomotion. The population-specific distribution and the different functional properties of the two ODH allozymes are most likely related to the different degree of tidal exposure which the two populations experience in nature. Only the slow allozyme possesses the regulatory properties which would allow a shift to the alternative anaerobic pathways utilized during these longer exposure periods.Abbreviations ODH octopine dehydrogenase - LDH lactate dehydrogenase - PHI phosphohexose isomerase