Article Figures & Data
Figures
- Fig. 1.
The time course plot of the product formation under anaerobic conditions using glucose oxidase to remove oxygen (as described in Materials and Methods) is presented for O6BG in hAO (A), O6BG in mAO (B), dantrolene in hAO (C), and dantrolene in mAO (D). Saturating concentration (five times the Km) of both oxidative (O6BG, 200 μM) and reductive (dantrolene, 30 μM) substrates were used for these kinetic assays. The kinetic models used in fitting these data sets are presented as blue insets, and the R2 values are mentioned on the bottom right side of each graph. The MAM was used for (A), and the Michaelis-Menten linear model was used for (B–D). Models were selected according to the kinetic trend of the product formation in each data set.
- Fig. 2.
(A) The nitro reduction pathway: six electrons are required for a full nitro reduction, but nitroso and hydroxylamine intermediates could also be formed during this process. (B) A schematic presentation of the anaerobic AO catalytic cycle for O6BG as the oxidative substrate and dantrolene as the reductive substrate. The electron flow pathway from the MoCo site to the FAD site through the two iron-sulfur clusters is presented with a dashed arrow. The electron leakage through nitroso and hydroxylamine intermediates formed as a result of incomplete nitro reduction can pose a risk of TDI of the enzyme (orange dotted arrow). However, this is not the case for O6BG oxidation by AO.
- Fig. 3.
The time course plot of the ADNTN formation under anaerobic conditions using a saturating amount of O6BG (200 μM) as the oxidative substrate and dantrolene as the reductive substrate (30 μM). A glucose oxidase system was used to remove oxygen (as described in Materials and Methods) in all of the anaerobic reactions. The reductive half-reaction remained linear and was fit to the Michaelis-Menten model in all the mutants, F885L (black, R2 = 0.98), V811A (magenta, R2 = 0.95), and FLVA (purple, R2 = 0.96), similar to hAO (red, R2 = 0.98) and mAO (blue, R2 = 0.91).
- Fig. 4.
The time course plot of 8-oxo-O6BG formation by hAO (A), mAO (B), F885L (C), V811A (D), and FLVA double mutant (E) under anaerobic conditions using saturating concentration of O6BG (200 μM) and dantrolene (30 μM) is presented here. The data for all the enzymatic reactions were fit to the MAM, except for mAO, for which the linear Michaelis-Menten model was used.
Tables
- TABLE 1
The human to monkey (hAO#mAO) mutants were made using the primers, DNA templates, and the sequencing primers as described
Plasmid (hAO#mAO) Primer DNA template Sequencing primer F885L FW: 5′-cagacccatttcaataactaacaggctttcatccagg-3′ pTHco-AOX1 5′-attgttgccagcacactgaa-3′ (nucleotides 2401–2800) RV: 5′-cctggatgaaagcctgttagttattgaaatgggtctg-3′ V811A FW: 5′-aataataccggttttcagcgctttaccaccaaatgcacc-3′ pTHco-AOX1 RV: 5′-ggtgcatttggtggtaaagcgctgaaaaccggtattatt-3′ FLVA FW: 5′-aataataccggttttcagcgctttaccaccaaatgcacc-3′ F885L RV: 5′-ggtgcatttggtggtaaagcgctgaaaaccggtattatt-3′ - TABLE 2
A comparison between the rate of product formation between the two species as well as the mutants under anaerobic conditions using a glucose oxidase system as described in the Materials and Methods section.
MAM was the model of choice in fitting the O6BG (200 μM) product formation for hAO and all the mutants and Michaelis-Menten linear model was used to fit the O6BG product formation in mAO as well as the product formation for dantrolene (30 μM) in both species and the mutants.
Rate of 8-oxo-O6BG formation Rate of ADNTN formation k3_ox k4 k5 k3_red min−1 min−1 min−1 min−1 hAO 110 ± 20.7 0.160 ± 0.0494 9.28 ± 2.03 0.659 ± 0.0182 mAO 2.19 ± 0.0696 — — 0.156 ± 0.00876 F885L 38.7 ± 3.85 0.0388 ± 0.0144 7.90 ± 2.71 0.701 ± 0.0193 V811A 40.7 ± 8.51 0.158 ± 0.0486 2.95 ± 0.522 0.336 ± 0.0143 FLVA 30.4 ± 5.02 0.0659 ± 0.0289 7.50 ± 1.37 0.486 ± 0.0199 - TABLE 3
The change in the ratio of the concentration of the oxidative to reductive product (OAR) with time under anaerobic conditions (as described in the Materials and Methods section) and saturating amount of the oxidative and reductive substrates, O6BG (200 μM) and dantrolene (30 μM), respectively
Time, min [8-oxo-O6BG]/[ADNTN] hAO mAO F885L V811A FLVA μM/μM μM/μM μM/μM μM/μM μM/μM 0.2 55.6 ± 19.3 4.41 ± 0.622 86.0 ± 42.2 46.1 ± 23.9 21.7 ± 4.54 3 71.8 ± 26.8 3.59 ± 1.38 39.8 ± 8.20 34.4 ± 9.70 28.8 ± 1.54 10 71.3 ± 16.9 4.43 ± 1.32 42.1 ± 7.22 39.8 ± 8.45 39.5 ± 2.71 20 61.0 ± 12.0 5.99 ± 0.881 47.2 ± 2.31 37.4 ± 8.09 40.3 ± 2.51 30 52.7 ± 5.68 5.41 ± 1.48 43.4 ± 1.34 33.8 ± 4.41 40.6 ± 3.53 45 39.3 ± 8.13 6.78 ± 1.64 38.6 ± 3.67 27.7 ± 2.54 38.1 ± 5.25 60 33.5 ± 5.94 7.63 ± 1.72 34.6 ± 3.24 23.9 ± 1.79 34.8 ± 6.57 90 25.3 ± 3.30 7.90 ± 1.60 26.7 ± 2.40 19.5 ± 1.09 29.5 ± 4.25 120 20.6 ± 2.57 8.53 ± 2.62 22.3 ± 2.71 16.3 ± 0.875 21.4 ± 0.972 Average 50.3 ± 20.8 5.83 ± 1.83 42.3 ± 18.2 31.0 ± 9.85 32.8 ± 7.70
