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Lactate oxidase


Official Full Name
Lactate oxidase
Background
In enzymology, a lactate 2-monooxygenase (EC 1.13.12.4) is an enzyme that catalyzes the chemical reaction: (S)-lactate + O2↔ acetate + CO2 + H2O. Thus, the two substrates of this enzyme are (S)-lactate and O2, whereas its 3 products are acetate, CO2, and H2O. This enzyme belongs to the family of oxidoreductases, specifically those acting on single donors with O2 as oxidant and incorporation of two atoms of oxygen into the substrate (oxygenases). The oxygen incorporated need not be derived from O with incorporation of one atom of oxygen (internal monooxygenases o internal mixed-function oxidases). This enzyme participates in pyruvate metabolism. It employs one cofactor, FMN.
Synonyms
EC 1.13.12.4; 9028-72-2; lactate oxidative decarboxylase; lactate oxidase; lactic oxygenase; lactate oxygenase; lactic oxidase; L-lactate monooxygenase; lactate monooxygenase; L-lactate-2-monooxygenase; lactate 2-monooxygenase

Catalog
Product Name
EC No.
CAS No.
Source
Price
CatalogEXWM-0617
EC No.EC 1.13.12.4
CAS No.9028-72-2
Source
CatalogNATE-0375
EC No.EC 1.13.12.4
CAS No.9028-72-2
SourcePediococcus sp.
CatalogDIA-208
EC No.EC 1.1.3.2
CAS No.
SourceMicroorganism
CatalogDIA-156
EC No.EC 1.13.12.4
CAS No.9028-72-2
SourceAerococcuss vir...
Related Services
Related Protocols
Lactate Oxidase -Enzymatic Assay Protocol
Related Reading

Lactic acid oxidase (Lox, EC 1.1.3.2) is a homotetrameric flavoprotein that catalyze the oxidation of α-hydroxyacids. The systematic name of this enzyme is (S)-lactate:oxygen 2-oxidoreductase (decarboxylating). Other names in common use include lactate oxidative decarboxylase, lactate oxidase, lactic oxygenase, lactate oxygenase, lactic oxidase, L-lactate monooxygenase, lactate monooxygenase, L-lactate-2-monooxygenase. L-lactate oxidase is a new member of the FMN-containing enzyme family, comprising glycolate oxidase, L-lactate oxidase, L-lactate monooxygenase, flavocytochrome b2, and long-chain α-hydroxyacid oxidase, and L-mandelate dehydrogenase.

Structure

In the crystal structure of the FMN enzyme, the co-factor isoalloxazine system almost always exists in a bent and therefore reduced form, but exists in the structure of thioredoxin in oxidized and reduced form. The reduced cofactor is regenerated by the peroxo adduct at C4 in the isoalloxazine-ring system. The structures of four related enzymes, glycolate oxidase (GLO), a soluble chimeric form of mandelate oxidase (MDH), long-chain hydroxy acid oxidase (HAO), and flavocytochrome b2 (B2) have been determined by X-ray crystallography. In these structures, each monomer contains a classic eight-stranded α/β-barrel, and the binding site of the FMN cofactor and substrate is located at the C-terminus of the β-barrel. In GLO and HAO, the biologically active unit consists of two tetramers, stacked slightly rotated. The active unit in MDH contains a tetramer, but in B2 it is a heterodimer containing two α/β-barrel with FMN-binding and substrate-binding sites. B2 has always been a research object to elucidate the reaction mechanism, but it is somewhat different from other compounds because one of the chains also contains a heme unit involved in electron transfer, so the regeneration of cofactors proceeds through a different pathway.

The asymmetric unit of lactate oxidase (LOX) contains two closely packed tetramers. Each tetramer forms a biologically active unit and has an internal 422 symmetry similar to glycolate oxidase. Each LOX tetramer measures approximately 50х100х100 Å and its C-terminus is folded to fill the central pore. Each LOX monomer superimposes well with each other, and the root mean square (r.m.s.) deviation is in the range of 0.19-0.69 Å. The most similar are C and F, and the most different are C and E. The following discussion is based on monomer A of LOX. The LOX tetramer was superimposed on GLO tetramer generated by crystallography with an r.m.s. deviation of 1.24 Å for 1268 Cα atom. The 374 amino acids in each LOX subunit are folded into an α/β-barrel with two short β-strands at the bottom of the barrel. One monomer contains 15 α-helixes of different sizes. The FMN binding site and active site are located at the C-terminus of the β-barrel, where LOX residues 190-220 form a lid-like structure of the active site. This region is poorly conserved in sequence alignment and is disordered in the GLO, HAO, and B2 structures.

The L-Lactate oxidase tetramer, describing half of the asymmetric unit. Figure 1. The L-Lactate oxidase tetramer, describing half of the asymmetric unit. (Leiros I. 2006)

The monomer structure of L-Lactate oxidase tetramer. Figure 2. The monomer structure of L-Lactate oxidase tetramer. (Leiros I. 2006)

Catalytic Mechanism

Lactate oxidase is one of the FMN-dependent enzymes, which catalyzes the conversion of α-hydroxy acids to α-keto acids through the two-electron reduction of FMN cofactors. Although the details of this mechanism are still being debated, it eventually leads to the transfer of two electrons and two protons from the reduced cofactor to molecular oxygen, which generates hydrogen peroxide and α-keto acid, and eventually produces oxidized cofactors. The reaction proceeds through a ping-pong mechanism, which has successive reduction and oxidation steps. The first step is to extract protons from the substrate α-carbon with adjacent bases to generate carbanions, and then transfer the two electrons to the cofactor.

Application

Lactate concentration has been widely used as a key parameter in clinical diagnostic to assess patient health and disease research and for continuous monitoring in the surgery, sports medicine, shock/trauma and food industry. The most commonly used analytical methods for the determination of lactate include high performance liquid chromatography (HPLC), fluorescence, colorimetric, chemiluminescence, and magnetic resonance spectroscopy. Although these methods provide results, they have disadvantages, such as the time-consuming requirement of sample preparation, costly due to the need for expensive machinery and trained manpower. However, biosensors can overcome these limitations. Compared with various methods that can be used for lactate detection, biosensing methods have the advantages of simplicity, directness and real-time, without sample preparation (may require sample dilution), combining fast response with high specificity, economy and efficiency. The concept of an enzyme-coupled biosensor electrode is to place the enzyme close to the electrode surface. The enzyme involved must catalyze a reaction that involves the consumption of an electroactive reactant or the generation of an electroactive substance. Then monitor the consumption or production process and directly measure the analyte concentration. In the manufacture of L-lactate biosensors, the most commonly used biological recognition elements are L-lactate dehydrogenase (LDH) and L-lactate oxidase (LOD). L-LOD catalyzes the oxidation of L-lactate to pyruvate in the presence of dissolved oxygen and forms hydrogen peroxide, which is electrochemically active and can be reduced or oxidized to give a proportional to the of L-lactate concentration, so it can be used in biosensors.

Basic principle of L-lactate oxidase biosensor. Figure 3. Basic principle of L-lactate oxidase biosensor. (Rathee K. 2015)

References

  1. Yorita, K., Janko, K., Aki, K., Ghisla, S., Palfey, B.A., Massey, V. On the reaction mechanism of L-lactate oxidase: quantitative structure-activity analysis of the reaction with para-substituted L-mandelates. Proc Natl Acad Sci U S A, 1997, 94(18):9590-5.
  2. Leiros, I., Wang, E., Rasmussen, T., Oksanen, E., Repo, H., Petersen, S.B., Heikinheimo, P., Hough, E. The 2.1 A structure of Aerococcus viridans L-lactate oxidase (LOX). Acta Crystallogr Sect F Struct Biol Cryst Commun, 2006, 62(Pt 12): 1185-90.
  3. Rathee, K., Dhull, V., Dhull, R., Singh, S. Biosensors based on electrochemical lactate detection: A comprehensive review. Biochem Biophys Rep, 2015, 5: 35-54.

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