Figure 1.
SDS-PAGE analysis of protein samples of PH_IND from E. coli BL21 (DE3).
Line 1. Whole cells of pET-28a(+); Line 2. Cell extracts of pET-28a(+); Line 3. Precipitation of pET-28a(+); Line 4. Whole cells of strain PH_IND; Line 5. Cell extracts of strain PH_IND; Line 6. Precipitation of strain PH_IND; M. Protein markers. Arrows show the positions of the six ORFs. ORF1. 10.4 kDa; ORF2. 37.6 kDa; ORF3. 10.5 kDa; ORF4. 59.2 kDa; ORF5. 13.5 kDa; ORF6. 38.6 kDa. SDS-PAGE was performed with 5% and 15% acrylamide concentrations for the concentrating and separating gels, respectively.
Table 1.
Characteristics of indigoids produced by whole cells of strain PH_IND.
Figure 2.
Identification of transformation products by TLC.
The transformation samples were extracted with equal volume of ethyl acetate and concentrated by N2. 200 µL of the extracts were applied to the TLC plates (silica gel 60 F254), and then the TLC plates were resolved with a solvent mixture of dichloromethane-methanol (50∶1, v/v). The samples were designated as following: 1. Indigo (standard); 2. Products of indole transformation; 3. Products of 4-methylindole transformation; 4. Products of 5-methylindole transformation; 5. Products of 7-methylindole transformation; 6. Products of 5-methoxyindole transformation; 7. Products of 4-chloroindole transformation; 8. Products of 7-chloroindole transformation. The products with different Rf values were indicated in three regions by the arrows.
Figure 3.
Homology modeling and identified substrate tunnel.
A. The residues involved in coordinating dinuclear iron. These residues were labeled in cyan, and the dinuclear iron sites were shown in sphere; B. Tunnel identified in the homology modeling of PHN component from Arthrobacter sp. W1 using CAVER. The white sticks represent five formed residues, i.e. Thr-201, Asn-202, Phe-205, Glu-231 and Met-235. The tunnel is labeled as yellow surface and the residues formed entrance are in purple.
Table 2.
Comparisons of active sites in various oxygenases.
Figure 4.
Interactions between PHNW1 component and indole derivatives.
Orientations of docked indoles in the active site of PHNW1 component: A. Indole; B. 4-Methylindole; C. 5-Methylindole; D. 7-Methylindole; E. 4-Chloroindole; F. 7-Chloroindole; G. 5-Methyoxyindole; H. 3-Methylindole. Atom designation: carbon atom, blue; hydrogen atom, white; chlorine atom, green; oxygen atom, red. Orange spheres represent for diiron, of which located above is designated as Fe1; green represent for tunnel entrance residues Asn-202 and Phe-205; other important residues are shown in grey.
Table 3.
Residues involved in binding different indoles.
Figure 5.
Summary of transformation pathways of indole by various oxygenases.
DO. dioxygenase; SO. styrene oxygenase; MO. monooxygenase. R represents for substitute group i.e. methyl-, chloro-, methyoxy-, etc. The pathways catalyzed by strain PH_IND could be proposed by C-3 oxidation and C-7 oxidation pathways. The indole is firstly hydroxylated at the C-3 positions to form indoxyl (3-hydroxyindole) by strain PH_IND, which undergoes further oxidation to form isatin as well as the indigoids precursors. Finally, two molecules of indoxyl polymerize to form indigo, and indoxyl can form indirubin with isatin. And the new compound is formed by 7-hydroxyindole and isatin.
Table 4.
Bacterial strains and plasmids used in this study.