Fig 1.
A segment of phylogenetic tree representing the relationship of ORF All2316 encoding Anabaena 7120 AKR to other closely related members of the AKR superfamily.
Analysis involved all annotated AKRs (155 proteins) that fall into 15 families of AKR superfamily along with the new AKR from Anabaena 7120. The complete tree and the sequence alignment of the entire AKR protein sequences are given in supporting information (S1 and S2 Files). Full names for the sequences used in this figure are available on the AKR homepage (http://www.med.upenn.edu/akr).
Fig 2.
Sequence alignment of Anabaena 7120 aldo- keto reductase All2316 and members of AKR13 family using PROMALS3D multiple sequence and structure alignment server (prodata.swmed.edu/promals3d/).
Representative sequences are colored according to predicted secondary structures (red: alpha-helix, blue: beta-strand). Consensus predicted secondary structure symbols: alpha-helix: h; beta-strand: e. Consensus amino acid symbols are: conserved amino acids are in bold and uppercase letters; aliphatic (I, V, L): l; aromatic (Y, H, W, F): @; hydrophobic (W, F, Y, M, L, I, V, A, C, T, H): h; alcohol (S, T): o; polar residues (D, E, H, K, N, Q, R, S, T): p; tiny (A, G, C, S): t; small (A, G, C, S, V, N, D, T, P): s; bulky residues (E, F, I, K, L, M, Q, R, W, Y): b; positively charged (K, R, H): +; negatively charged (D, E):-; charged (D, E, K, R, H): c.
Fig 3.
Relative normalized expression of AKR17A1 in Anabaena 7120 exposed to butachlor, cadmium, UV-B, heat, salt, drought and arsenic.
Transcript levels were determined by qRT-PCR. The 16S rRNA gene was used as an internal control for normalizing the variations in cDNA amounts. Biological triplicates were averaged. Bars indicate SE.
Fig 4.
Spot assay of E. coli strain BL21 (DE3) transformed with vector alone (BL/pET21a) and recombinant plasmid (BL/ pET21a-AKR) under different abiotic stresses.
3 μL of sequential dilutions (only growth at dilution level 10−2 is shown here) were spotted on the Luria Bertani (LB) plates supplemented with varying concentration of (a) cadmium, (b) mannitol, (c) arsenic and (d) NaCl. For (e) UV-B and (f) heat (50°C),cells were pretreated for different time intervals and then spotted on the LB plates. All spot tests were performed in triplicate.
Fig 5.
Growth behavior of E. coli BL21 (DE3) cells transformed with pET21a and pET21a-AKR in response to (a) cadmium (0.3 mM) (b) mannitol (600 mM), (c) heat (50°C), (d) NaCl (600 mM), (e) UV-B (10 min) and (f) arsenic (6 mM) stress using liquid culture assay.
The mean of three independent replicates is plotted with error bars indicating standard deviations. (a-f) represents the growth curves of E. coli strains in LB medium supplemented with 100 μg ml−1 ampicillin and 0.2 mM IPTG.
Fig 6.
Specific activity of recombinant AKR17A1 on diverse substrates.
Data represents the average of three measurements and bars indicate ±SD.
Table 1.
Steady-state kinetic parameters of recombinant His-tagged AKR17A1.
Kinetic constants are mean values of three independent experiments. Values in parentheses indicate standard error of the mean (SEM). The activity of AKR17A1 at various concentrations of substrates tested is given as Fig C in S3 File.
Fig 7.
Absorption spectrograms showing degradation of butachlor by NADPH dependent AKR17A1.
The analysis was performed in triplicates. The absorption spectra for each samples was recorded in 1 mL sample volume containing 50 mM potassium phosphate buffer (pH 7.4), 10 mM β-mercaptoethanol, 0.1 mM butachlor and either 0.12 mM NADPH, 5μg AKR or both.
Table 2.
Percent composition of butachlor and metabolites as determined by GC-MS analysis.
The GC-MS measurements were carried out with the metabolites extracted from the reaction mix containing 50mM potassium phosphate buffer pH 7.4, 0.4 mM butachlor and 10mM 2-mercaptoethanol, (1) neither NADPH nor AKR, (2) NADPH (0.12mM), (3) AKR (5 μg), or (4) both NADPH (0.12mM) and AKR (5 μg).
Fig 8.
GC charts of the reaction products of AKR17A1 with butachlor used as substrate.
(a) Control assay devoid of enzyme AKR17A1 and cofactor (NADPH); (b) reaction with NADPH alone; (c) enzymatic reaction with AKR17A1 alone; and (d) enzymatic reaction with both AKR17A1 and cofactor NADPH.