The bacterial-like lactate shuttle components from heterotrophic Euglena gracilis

The structural and kinetic analyses of the components of the lactate shuttle from heterotrophic Euglena gracilis were carried out. Mitochondrial membrane-bound, NAD(+)-independent D-lactate dehydrogenase (D-iLDH) was purified by solubilization with CHAPS and heat treatment. The active enzyme was a 62-kDa monomer containing non-covalently bound FAD as cofactor. D-iLDH was specific for D-lactate and it was able to reduce quinones of different redox potential values. Oxalate and L-lactate were mixed-type inhibitors of D-iLDH. Mitochondrial L-iLDH also catalyzed the reduction of quinones, but it was inactivated during the extraction with detergents. Both L-iLDH and D-iLDH were inhibited by the specific flavoprotein-inhibitor diphenyleneiodonium, suggesting that L-iLDH was also a flavoprotein. Affinity chromatography revealed that the E. gracilis cytosolic fraction contained two types of NAD+-dependent LDH specific for the generation of D- and L-lactate (D-nLDH and L-nLDH, respectively). These two enzymes were tetramers of 126-132 kDa and showed an ordered bi-bi kinetic mechanism. Kinetic properties were different in both enzymes. Pyruvate reduction by D-nLDH was inhibited by its two products; the D-lactate oxidation was 40-fold lower than forward reaction. L-lactate oxidation by L-nLDH was not detected, whereas pyruvate reduction was activated by fructose-1, 6-bisphosphate, K+ or NH4+. Interestingly, membrane-bound L- and D-lactate dehydrogenases with quinone reductase activity have been only detected in bacteria, whereas the activity of soluble D-nLDH has been identified in bacteria and some yeast. Also, FBP-activated L-nLDH has been found solely in lactic bacteria. Based on their similar kinetic and structural characteristics, a possible common origin among bacterial and E. gracilis lactic dehydrogenase enzymes is discussed. (c) 2005 Elsevier B.V. All rights reserved.