FMN-dependent oligomerization of putative lactate oxidase from Pediococcus acidilactici

Abstract

Lactate oxidases belong to a group of FMN-dependent enzymes and they catalyze a conversion of lactate to pyruvate with a release of hydrogen peroxide. Hydrogen peroxide is also utilized as a read out in biosensors to quantitate lactate levels in biological samples. Aerococcus viridans lactate oxidase is the best characterized lactate oxidase and our knowledge of lactate oxidases relies largely to studies conducted with that particular enzyme. Pediococcus acidilactici lactate oxidase is also commercially available for e.g. lactate measurements, but this enzyme has not been characterized in detail before. Here we report structural characterization of the recombinant enzyme and its co-factor dependent oligomerization. The crystal structures revealed two distinct conformations in the loop closing the active site, consistent with previous biochemical studies implicating the role of loop in catalysis. Despite the structural conservation of active site residues, we were not able to detect either oxidase or monooxygenase activity when L-lactate was used as a substrate. Pediococcus acidilactici lactate oxidase is therefore an example of a misannotation of an FMNdependent enzyme, which catalyzes likely a so far unknown oxidation reaction.

Monitoring of Lactate in Interstitial Fluid, Saliva and Sweat by Electrochemical Biosensor: The Uncertainties of Biological Interpretation

“…The reported Aerococcus viridans enzyme K m ranges between 0.104 mM [33] and 0.94 mM [34], though the majority of reports indicate a value around 0.7 mM [35][36][37][38]. Surprisingly, despite the greater use of Pediococcus sp enzyme, there is only one reported K m for this source of 0.52 mM [39]. For the crosslinked Pediococcus sp in this study, based on half maximum current in the calibration plots (Figure 3), the K m would appear to be between 0.6-0.8 mM.…”

Flavin Mononucleotide-Dependent l-Lactate Dehydrogenases: Expanding the Toolbox of Enzymes for l-Lactate Biosensors

“…The results showed a single major SEC elution peak for all samples and masses that are 3.5 and 4.0 times the theoretical mass of the monomers for Ss LDH and Ea LDH, respectively, indicating tetrameric structures for these two enzymes. A tetrameric oligomerization, determined by SEC, has also been reported for Pa LCTO . Elution peaks of Lj LDH and Lh LDH displayed 7.5 and 7.7 times the theoretical masses of their monomers, respectively, implying that they form octameric structures instead of tetramers.…”

“…The N-terminal purification tag (-GSS-HHHHHH-G-LEVLFQGP-) was added between the start-Met and the second amino acid according to the protocol of Gibson Assembly (New England Biolabs) using 146 and 110 bp overlaps at the 5′ and 3′ end, respectively. The native form of Pa LCTO was cloned in a pNIC-CH vector as described previously . Predicted bacterial signal peptides of Pau LDH and Ss LDH were removed by amplifying their plasmids without the signal-peptide-coding region by PCR and re-ligating the linearized plasmid using the KLD Enzyme Mix (New England Biolabs).…”

“…Homogeneity of the enzymes was assessed by SDS-PAGE, and protein concentrations were calculated from their absorbance at 280 nm assuming theoretical extinction coefficients as determined by the ExPASy tool ProtParam from the amino acid sequence (48,360, 41,830, 24,870, 29,910, 28,420, 25,900, and 51,340 M –1 cm –1 for Lj LDH, Lh LDH, Ss LDH, Gb LDH, Ea LDH, Pa LCTO, and AvLOx, respectively). Purified Pa LCTO was produced as described previously using E. coli BL21 (DE3) expression, His-tag purification followed by cleavage of the tag by TEV protease digestion and size exclusion chromatography.…”

“…Commonly, the protein monomer of LOx has a mass of 39–44 kDa and shows a β 8 α 8 TIM-barrel fold, which is typical for the HAOx family. LOx is active as a homotetramer with the FMN cofactor noncovalently bound to each subunit. , The active site is formed by the isoalloxazine ring of FMN together with several well conserved substrate-binding residues at the edge of the β-barrel, and it is covered by the so-called “active-site lid” . This lid has been shown to be highly dynamic and to regulate substrate entry and product exit to and from the active site. , Even though the loop forming the lid can be found in all members of the HAOx family, it is poorly conserved across the family, and due to its flexibility, it is usually disordered in HAOx crystal structures.…”