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

Abstract

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 ␣-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-␣-hydroxyacids, ␣-hydroxy-n-butyric acid, ␣-hydroxy-n-valeric acid, etc., and also the aromatic ␣-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 pK a 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.␣-Hydroxyacid oxidizing enzymes, a family of flavoprotein enzymes, share a series of common characteristics. Within the family, the crystal structures of glycolate oxidase and flavocytochrome b 2 have been solved by x-ray diffraction studies (1-4). The reaction mechanisms of L-lactate monooxygenase from Mycobacterium smegmatis and flavocytochrome b 2 from Saccharomyces cerevisiae have been studied extensively (5, 6). L-Lactate monooxygenase utilizes L-lactate and other L-␣-hydroxyacids but is unique in accelerating the oxidative decarboxylation of the products, pyruvate (or corresponding keto acid) and hydrogen peroxide, to acetic acid (or corresponding carboxylic acid), carbon dioxide, and water. New members of this flavoenzyme family have been described including those utilizing aromatic and bulky substrates like mandelate and long chain ␣-hydroxyacids (7-9).We are studying one of the new enzymes of this family, L-lactate oxidase from Aerococcus viridans (10). This enzyme utilizes L-lactate and oxygen as L-lactate monooxygenase does but forms pyruvate and hydrogen peroxide as the final products instead of catalyzing the oxidative decarboxylation reaction. We believe that lactate oxidase is an ideal enzyme for studying the reaction mechanism of this enzyme family because of its considerable stability, and it is also a useful enzyme for the construction of a lactate sensor for biological applications.By analogy with the crystal structures of glycolate oxidase and flavocytochrome b 2 , it has been pointed out that glycine 99 in L-lactate monooxygenase is unique in the family of enzymes (except now mandelate dehydrogenase (11) also with glycine), because the other family members have an alanine residue at the homologous position (6). This residue, in glycolate oxidase and flavocytochrome b 2 , is in close contact with the flavin N-5 position on the re-face of the flavin. It was considered possible that the s...

The Ala95‐to‐Gly substitution in Aerococcus viridansl‐lactate oxidase revisited – structural consequences at the catalytic site and effect on reactivity with O2 and other electron acceptors

“…The grey line shows the spectrum after reaction for 200 ms. Inset shows the time-resolved absorption trace at 530 nm, normalized on the maximum absorption observed at this wavelength in the reaction. According to previous studies of avLOX [9,21], the change in the 530-nm band is a result of the formation and decay of a complex between reduced enzyme and pyruvate, and the inset shows the dynamics of the complex. .…”

“…A naturally occurring Ala/Gly residue variation within strand b1 is a prominent factor of structural variability around the FMN N5 atom [3][4][5][6][7]. Evidence from sitedirected mutagenesis reveals that Ala/Gly residue exchange in different enzymes impacts significantly upon the O 2 reactivity, in addition to having a major effect on the a-hydroxy acid substrate specificity [19][20][21][22][23]. However, the results also show that the Ala/Gly pattern does not constitute a simply addressable oxidase-dehydrogenase switch in a-hydroxy acid-oxidizing flavoenzymes, unlike vanillyl-alcohol oxidases where a similar pattern of Ala and Gly (or Pro) was proposed to determine flavoenzyme activity as a dehydrogenase and oxidase, respectively [24].…”

“…In the present study, we have focused on the LOX from Aerococcus viridans (avLOX), a prototypical oxidase-type member of the a-hydroxy acid-oxidizing flavoenzyme family that has been well characterized biochemically [9,21,25,26] and structurally [6,[27][28][29]. The enzyme is biotechnologically important for its commercialized use in analytical devices for blood L-lactate determination [30,31].…”

“…However, avLOX is also able to replace O 2 by another oxidant such as 2,6-dichlorophenol-indophenol (DCIP), which is exploited in the fabrication and application of L-lactate test strips [32]. An A95G variant of avLOX was previously constructed and shown to exhibit approximately 10-fold lower O 2 reactivity than the wild-type at 4°C [21]. The mutation was also shown to enhance the enzyme's specificity for long chain and generally larger sized a-hydroxy-acid substrates [21,25].…”

“…An A95G variant of avLOX was previously constructed and shown to exhibit approximately 10-fold lower O 2 reactivity than the wild-type at 4°C [21]. The mutation was also shown to enhance the enzyme's specificity for long chain and generally larger sized a-hydroxy-acid substrates [21,25].…”