Identification and Characterization of a Novel Three Component Salicylate Oxygenase from Sphingomonas yanoikuyae B1

1. Identification and Characterization of a Novel Three Component Salicylate Oxygenase from Sphingomonas yanoikuyae B1 Environmental Microbiology Laboratory Cho, Ok young

2. orf0 nahD bphA1c A3 bphC xylX xylY orf1 bphA1d bphK xylF XylE xylG xylJ xylQ xylK xylI H bphR bphA4 xylL nahE bphA1e bphX The nucleotide sequence encoding genes for aromatic hydrocarbon degradation from S. yanoikuyae B1 0 10,850 A2c 10,851 20,000 20,001 29,200 xylC bphB xylA xylM A2b bphA1b A2a bphA1a 29,201 38,958

3. XylMA H2O O2 Reductase (red) Ferredoxin (ox) ISP (red) NAD+ NADH + H+ NAD+ ISP (ox) Ferredoxin (red) Reductase (ox) • Aromatic ring dioxygenase NADH + H+ • Aromatic ring monooxygenase

4. Salicylate hydroxylase • - is a member of aromatic ring oxygenase transforming salicylate into catechol, • - is divided into at least two distinct enzyme groups. •  Pseudomonas putida PpG7 (plasmid NAH7) • - 45 kDa, monomeric flavoprotein (one mole of FAD) •  Pseudomonas cepacia • - two moles of FADs and two identical subunits(91 kDa) • (You et al, 1990) • Salicylate 5-hydroxylase from Ralstonia sp. Strain U2 has three component • oxygenase system • ;NsgGH (oxygenase component), NagAa(reductase), and NagAb (ferredoxin) NADH + H+ NAD+ O2 H2O + CO2

5. salicylate catechol Growth characteristics of growth B1 and EK504 on aromatic compounds m-toluate m-xylene Biphenyl Phenanthrene Naphthalene + + + + + B1 - + + + + EK504 (Adapted from Kim and Zylstra, 1999) nahD naphthalene cis-naphthalene dihydrodiol 2-hydroxy-chromene- 2-carboxylate trans-o-hydroxylbenzylidene pyruvic acid

6. 3kb B B E E E K Bg S E E X H Complementation of EK504 EK504 Km Nap Sal - - EK504 B B bphA1cA2c bphC orf1 nahD bphA3 + + pGJZ1553 EK504(pGJZ1553) 1kb B H - - EK504(pKEB1401) pKEB1401 pKEB1402 EK504(pKEB1402) S X + + pKEB1404 H X EK504(pKEB1404) The dashed arrow indicates the direction of transcription as determined by the nucleotide sequence. Abbreviations : B, BamHI; Bg, BglII; E, EcoRI; H, HindIII; K, KpnI; S, SstI; X, XbaI;

7. Growth of S. yanoikuyae B1 and EK504 on (methyl)salicylates as a sloe carbon source and energy sources a: A plus sign indicates the formation of a 1.0-mm-diameter colony within 2 days. b: A minus sign denotes the compound could not serve as sole carbon and energy source.

8. . Construction of an expression system for an ISP gene(bphA1cA2c) : The genes for an ISP(bphA1cA2c) and a ferredoxin(bphA3) were amplified by PCR, and then ligated into pCR T7/CT- TOPO vector for the expression in E. coli. 1. PCR amplication 5’ ATGTCGAACAAATTGCGC 3 ‘ 5’ AAGCCCTGGCGTGCTACT 3’ A3 A2c A1c 2. Ligation into a pCR T7/CT-TOPO vector 3. Transformation into E. coli BL21 (DE3) II. Characterization of bphA1cA2c 1. Resting cell assay 2. Structural identification of metabolites -Gas chromatography-mass spectrometry(GC- MS), - High pressure liquid chromatography (HPLC)

9. Transformation of salicylate into catechol by the expression system Formation of catechol from salicylate by E. coli BL21(DE3)(pKEB1570) containing bphA1cA2cA3. Spectra of 1 ml cleaved samples were recored at 3 hour intervals (0, 3, 6, and 9 h) by spectrophotometer.

10. Structural identification of metabolites formed during the incubation of recombinent E. coli strains in the presence of 3-methyl salicylate, 4-methyl salicylate and 5-methyl salicylate a (min) on GC Compound Rt Prominent ions (m/z, relative intensity %) 3-methylcatechol 12.82 268 (M+, 31), 253 (9), 180 (9), 165 (6), 149 (5), 73 (100) 3MS metabolite 12.85 268 (M+, 39), 253 (12), 180 (10), 165 (7), 149 (5), 73 (100) 4-methylcatechol 12.65 268 (M+, 44), 253 (7), 180 (9), 165 (5), 149 (5), 73 (100) 4MS metabolite 12.65 268 (M+, 41), 253 (7), 180 (7), 165 (5), 149 (5), 73 (100) 5MS metabolite 12.65 268 (M+, 48), 253 (8), 180 (10), 165 (5), 149 (5), 73 (100) a Rt ; Retentiontime

11. Salicylate biotransformation  BphA1CA2C BphA1CA2C 3300 nmol/min

12. 3-methyl salicylate biotransformation BphA1CA2C 2611 nmol/min

13. 4-methyl salicylate biotransformation 3277 nmol/min BphA1CA2C

14. 5-methyl salicylate biotransformation 2666 nmol/min BphA1CA2C

15. 27.89 Height Biodegradation of I-hydroxy 2-naphthoic acid by BphA1CA2C Time 0 4.69 Time 5.5hr

16. Time15hr

17. Tests to confirm assocation of BphA1cA2c with common ferredoxin and reductase EK385 nahD bphA1c bphA2c bphA3 bphC Complementation of EK385 on Salicylate Km + ? pGJZ1553 pKEB1402 MB1 bphR bphA4 Complementation of MB1 on Bph m-Xyl m-Tol Nap phe Sal + + + - + + Cm pKEB1105

18. Proposed pathway for naphthalene catabolism by Sphingomonas yanoikuyae B1 NADH + H+ NAD+ TCA cycle Reductase BphA4 catechol naphthalene Ferredoxin BphA3 Salicylate Oxygenase BphA1cA2c H2O O2 Dioxygenase O2 cis- 1,2- dihydro-1,2- dihydroxynaphthalene salicylate

19. Conclusion * Complementation and metabolite analysis experiments demonstrated that BphA1cA2c is a novel three component oxygenase converting salicylate into catechol. * BphA1cA2c also oxidized methyl salicylates into corresponding catechols. * BphA1cA2c are associated with a common ferredoxin (BphA3) and a reductas (BphA4) in constituting electron transfer system. * BphA1cA2c from S. yanoikuyae B1 does not belong to the major family of the known salicylate hydroxylases. Furthermore, it constitutes a new oxygenase subfamily.

20. Further works * Complementation tests: EK504, EK385 * Test whether BphA1cA2c work together with BphA3A4 more efficiently in degrading aromatic acids * Biodegradation test of various substrates by BphA1CA2CA3 • 1- hydroxy 2 naphthoic acid • chloro benzoate, benzoate, toluate ….