Kenji Aki

L-lactate oxidase and L-lactate monooxygenase: Mechanistic variations on a common structural theme

Properties of L-lactate oxidase from Aerococcus viridans are described. The gene encoding the enzyme has been isolated. From its cDNA sequence the amino acid sequence has been derived and shown to have high similarity with those of other enzymes catalyzing oxidation of L-alpha-hydroxy acids, including flavocytochrome b2, lactate monooxygenase, glycolate oxidase, mandelate dehydrogenases and a long chain alpha-hydroxy acid oxidase. The enzyme is expressed in Escherichia coli, and is a flavoprotein containing FMN as prosthetic group. It shares many properties of other alpha-hydroxy acid oxidizing enzymes, eg stabilization of the anionic semiquinone form of the flavin, facile formation of flavin-N(5)-sulfite adducts and a set of conserved amino acid residues around the bound flavin. Steady-state and rapid reaction kinetics of the enzyme have been studied and found to share many characteristics with those of L-lactate monooxygenase, but to differ from the latter in quantitative aspects. It is these quantitative differences between the two enzymes which account for the differences in the overall reactions catalyzed. These differences arise from different stabilities of a common intermediate of reduced flavin enzyme and pyruvate. In the case of the monooxygenase this complex is very stable and is the form that reacts with O2 to give a complex in which the oxidative decarboxylation occurs, yielding the products, acetate, CO2, and H2O (Lockridge O, Massey V, Sullivan PA (1972) J Biol Chem 247, 8097-8106). With lactate oxidase, the complex dissociates rapidly, with the result that it is the free reduced flavin form of the enzyme that reacts with O2, to give the observed products, pyruvate and H2O2.

Transaminases of branched chain amino acids: IV. Purification and properties of two enzymes from rat liver

Abstract Two transaminases for the branched chain amino acids (valine, leucine and isoleucine) with α-ketoglutarate were isolated from normal rat liver and purified by DEAE-cellulose, hydroxylapatite and Sephadex column chromatographies. One enzyme (enzyme I) was eluted by 0.02 M phosphate buffer from a DEAE-cellulose column and catalyzed the transamination of all three amino acids. The other enzyme (enzyme II) was eluted by 0.18 M buffer and was specific for leucine. Half the total activity of enzyme I was found in the supernatant, while only one quarter of the activity of enzyme II was localized in the supernatant. The rest of the total activity remained in the mitochondrial fraction. The optimal pH of enzyme I was 8.2 and that of enzyme II was 8.7. The K m value for leucine of enzyme I was 7.5·10 −4 M and that for enzyme II was 2.5·10 −2 M. Enzyme I was activated by 2-mercaptoethanol but enzyme II was not. Anti-serum against hog heart transaminase, which has similar properties to rat liver enzyme I, inhibited the activity of enzyme I but not that of enzyme II. The two enzymes were compared and their physiological significance discussed.

Functional association between nicotinic acetylcholine receptor and sarcomeric proteins via actin and desmin filaments

By affinity chromatography utilizing α‐cobrotoxin from digitonin‐solubilized fractions of rabbit skeletal muscle, we found that many proteins are associated with the nicotinic acetylcholine receptor (AChR). In addition to the proteins we previously reported to bind to AChR (including dystrophin‐dystrophin‐associated protein (DAP) complex, utrophin, rapsyn, and actin; Mitsui et al. [1996] Biochem. Biophys. Res. Commun.224:802–807), α‐actinin, desmin, myosin, tropomyosin, troponin T, and titin are also identified to be associated with AChR. Alkaline treatment or Triton X‐100 solubilization released dystrophin‐DAP complex, utrophin, and rapsyn from the AChR fraction, while actin and desmin remained associated. These findings demonstrate that AChR is supported primarily by a submembranous organization of actin and desmin filaments, and is linked to sarcomeric proteins via these filaments. To further investigate whether the association has any functional role, we studied the effect of acetylcoline on ATPase activity of the AChR fraction. Acetylcholine (0.5–4 μM) significantly activated Mg2+‐ATPase activity of digitonin‐solubilized AChR fraction (P < 0.05). Furthermore, we found that desmin as well as actin activated myosin Mg2+‐ATPase activity. From these findings, it is suggested that desmin and actin form a submembranous organization in the postsynaptic region, and function as mediators of excitation of AChR to the sarcomeric contraction system. J. Cell. Biochem. 77:584–595, 2000.

Conversion of L-Lactate Oxidase to a Long Chain α-Hydroxyacid Oxidase by Site-directed Mutagenesis of Alanine 95 to Glycine

A mutant form of L-lactate oxidase (LOX) from Aerococcus viridans in which alanine 95 was replaced by glycine was constructed as a mimic of L-lactate monooxygenase but proved instead to be a mimic of the long chain α-hydroxyacid oxidase from rat kidney. A95G-LOX keeps oxidase activity with L-lactate at the same level as wild type LOX but has much enhanced oxidase activity with longer chain L-α-hydroxyacids, α-hydroxy-n-butyric acid, α-hydroxy-n-valeric acid, etc., and also the aromatic α-hydroxyacid, L-mandelic acid. Kinetic analysis of the activity with these substrates indicates that the reduction of the enzyme bound flavin by substrates is the rate-limiting step in A95G-LOX. The affinity of pyruvate for the reduced enzyme is increased, and sulfite binding to the oxidized enzyme is weaker in A95G-LOX than in native enzyme. Wild type LOX stabilizes both the neutral and anionic flavin semiquinones with a pKa of 6.1, but A95G LOX stabilizes only the anionic semiquinone form. These results strongly suggest that the environment around the N5-C4a region of the flavin isoalloxazine ring is changed by this mutation.

Mitochondrial damage in patients with long-term corticosteroid therapy: development of oculoskeletal symptoms similar to mitochondrial disease

Abstract. Two patients with long-term corticosteroid administration sporadically developed limb muscle wasting followed by ophthalmoplegia, and the skeletal muscle pathology revealed ragged-red fibers (RRFs) with abnormal mitochondria, in addition to the findings of corticosteroid myopathy. The oculoskeletal symptoms of the present cases resemble those of chronic progressive external ophthalmoplegia, a type of mitochondrial disease. The ocular muscles have more RRFs than limb muscles, and large multiple deletions of mitochondrial DNA was detected in ocular and limb muscles of the two patients by PCR but not by Southern blotting. Immunohistochemistry demonstrated that 8-hydroxy-deoxyguanosine (8-OH-dG) and 4-hydroxy-2-nonenal were intensely stained in skeletal muscles of these patients particularly in RRFs. High-performance liquid chromatography with electrochemical detection analysis revealed an increase in 8-OH-dG from mitochondrial DNA. These findings may suggest that long-term corticosteroid administration potentially induces oxidative stress-mediated mitochondrial damage, resulting in the development of the oculoskeletal symptoms in some patients.

Superoxide-mediated release of iron from ferritin by some flavoenzymes

NADH-lipoamide dehydrogenase mobilized iron from ferritin under aerobic conditions. Superoxide dismutase strongly inhibited this mobilization, indicating that the superoxide radical is generated by the enzymatic reaction and release iron from ferritin. Addition of lipoamide as an electron acceptor to NADH-lipoamide dehydrogenase increased the release of iron from ferritin and this release was partially inhibited by superoxide dismutase. Similarly, addition of menadione (2-methyl-1, 4-naphthoquinone) as an electron acceptor to xanthine-xanthine oxidase promoted the release of iron from ferritin and this release was strongly inhibited by superoxide dismutase. These results suggest that dihydrolipoamide and semiquinone of menadione can react with oxygen to form the superoxide radical that mediates release of iron from ferritin.

Formation of a complex between yeast l-lactate dehydrogenase (cytochrome b2) and cytochrome c: Ultracentrifugal and gel chromatographic analyses

Yeast L-lactate dehydrogenase formed a stable complex with cytochrome c in weakly alkaline solution of low ionic strength. The binding ratio of cytochrome c to the enzyme depended on whether free cytochrome c was present: In the presence of a micromolar concentration of cytochrome c the enzyme formed a complex with about two molecules of cytochrome c, whereas the enzyme was in a 1:1 molecular complex after removal of free cytochrome c. This suggests that the binding of one molecule of cytochrome c changes the affinity of the other binding site on the enzyme for cytochrome c. The enzyme consists of four presumably identical subunits, each containing a binding site for cytochrome c. Thus, present data confirm the concept of negative cooperativity between the subunits of the enzyme molecule in their interaction with cytochrome c.

L-lactate oxidase from Aerococcus viridans crystallized as an octamer. Preliminary X-ray studies

Crystals of flavoenzyme L-lactate oxidase from Aerococcus viridans (LOX) have been obtained that diffract to 3.0 A resolution (P2(1)2(1)2(1), a = 118.4 A, b = 138.4 A, c = 194.6 A). Crystallographic studies suggest that the enzyme may exist as an octameric form with non-crystallographic two- and four-fold axes in the center of the octamer. The four-fold axis makes the tetramer tight, and the tetramers lie upon one another by the two-fold axis.

Sodium-induced aggregation of phosphatidic acid and mixed phospholipid vesicles

Sodium-induced aggregations of sonicated vesicles prepared from synthetic phosphatidic acid and from its 1:1 mixtures with synthetic phosphatidylethanolamine and phosphatidylcholine were studied by turbidimetric measurements. The aggregation reactions were almost completely reversible on change in the Na+ concentration, pH or temperature. The threshold concentrations of Na+ for aggregations of pure dipalmitoylphosphatidic acid vesicles and mixed dipalmitoylphosphatidylethanolamine- and dimyristoylphosphatidylcholine-dipalmitoylphosphatidic acid vesicles were found to be 200, 310 and 550 mM, respectively, at 25 degrees C and pH 7.2. The hydrocarbon chain lengths of phosphatidic acid and phosphatidylethanolamine had little effect on the threshold concentrations. The threshold concentrations for phospholipid vesicles composed of phosphatidic acid alone or its 1:1 mixture with phosphatidylethanolamine were changed by varying either the pH or temperature, while that for phosphatidylcholine-phosphatidic acid vesicles was almost independent of the pH and temperature, implying that aggregation of the latter vesicles is induced by a somewhat different mechanism.

Structural basis of free reduced flavin generation by flavin reductase from Thermus thermophilus HB8

Background: TTHA0420 is a flavin reductase, which makes free reduced flavin involved in a variety of fields. Results: We determined the dual binding mode of the substrate and co-factor flavins of TTHA0420. Conclusion: A specific motif YGG in the C terminus functions to regulate the alternative binding of NADH and substrate flavin. Significance: Our results have mechanistic implications for the reductase with two flavins. Free reduced flavins are involved in a variety of biological functions. They are generated from NAD(P)H by flavin reductase via co-factor flavin bound to the enzyme. Although recent findings on the structure and function of flavin reductase provide new information about co-factor FAD and substrate NAD, there have been no reports on the substrate flavin binding site. Here we report the structure of TTHA0420 from Thermus thermophilus HB8, which belongs to flavin reductase, and describe the dual binding mode of the substrate and co-factor flavins. We also report that TTHA0420 has not only the flavin reductase motif GDH but also a specific motif YGG in C terminus as well as Phe-41 and Arg-11, which are conserved in its subclass. From the structure, these motifs are important for the substrate flavin binding. On the contrary, the C terminus is stacked on the NADH binding site, apparently to block NADH binding to the active site. To identify the function of the C-terminal region, we designed and expressed a mutant TTHA0420 enzyme in which the C-terminal five residues were deleted (TTHA0420-ΔC5). Notably, the activity of TTHA0420-ΔC5 was about 10 times higher than that of the wild-type enzyme at 20–40 °C. Our findings suggest that the C-terminal region of TTHA0420 may regulate the alternative binding of NADH and substrate flavin to the enzyme.