Insights into the decarboxylative hydroxylation of salicylate catalyzed by the Flavin-dependent monooxygenase salicylate hydroxylase

  • Xiya Wang
  • Qianqian Hou
  • Yongjun Liu
Regular Article


Salicylate hydroxylase (SALH) is a Flavin-dependent monooxygenase responsible for the transformation of salicylate to catechol. In this article, on the basis of the crystal structure obtained from Pseudomonas putida S-1, we performed combined quantum mechanical/molecular mechanical (QM/MM) calculations to investigate the reaction mechanism of SALH. Since the formation of C4a-hydroperoxyflavin has been theoretically proven to be a barrierless process, our calculations started from the C4a-hydroperoxyflavin intermediate. The whole enzymatic reaction contains two parts: the hydroxylation and decarboxylation. Our calculation results indicate that the deprotonated substrate is the active form, whereas the neutral form of salicylate corresponds to very a high energy barrier (39.8 kcal/mol) for the hydroxylation process, which is in line with the experimental result that the optimum pH is 7.6. The calculated results with the deprotonated substrate indicate that the hydroxylation and decarboxylation occur in a stepwise manner and the decarboxylation process is calculated to be the rate-limiting step with an energy barrier of 14.5 kal/mol. Calculations using different functionals (B3LYP, BP, BVWN, PBE, M06 and TPSSH) suggest that the catalytic reaction is highly exothermic, which is consistent with its similar enzyme (PHBH).


Salicylate hydroxylase (SALH) Monooxygenase Hydroxylation Decarboxylation QM/MM method 



This work was supported by the National Natural Science Foundation of China (21773138, 21403210).

Compliance with ethical interest

Conflict of interest

The authors declare no competing financial interest.

Supplementary material

214_2018_2278_MOESM1_ESM.docx (721 kb)
Supplementary material 1 (DOCX 721 kb)


  1. 1.
    Zhao Y, Christensen SK, Fankhauser C, Cashman JR, Cohen JD, Weigel D, Chory J (2001) A role for flavin monooxygenase-like enzymes in auxin biosynthesis. Science 291(5502):306–309CrossRefPubMedGoogle Scholar
  2. 2.
    Macchiarulo A, Camaioni E, Nuti R, Pellicciari R (2009) Highlights at the gate of tryptophan catabolism: a review on the mechanisms of activation and regulation of indoleamine 2, 3-dioxygenase (IDO), a novel target in cancer disease. Amino Acids 37(2):219–229CrossRefPubMedGoogle Scholar
  3. 3.
    Hofrichter M (2002) Review: lignin conversion by manganese peroxidase (MnP). Enzym Microb Technol 30(4):454–466CrossRefGoogle Scholar
  4. 4.
    Meneely KM, Lamb AL (2007) Biochemical characterization of an FAD-Dependent monooxygenase, the ornithine hydroxylase from Pseudomonas aeruginosa, suggests a novel reaction mechanism. Biochemistry 46(42):11930–11937CrossRefPubMedPubMedCentralGoogle Scholar
  5. 5.
    Colonna S, Gaggero N, Pasta P, Ottolina G (1996) Enantioselective oxidation of sulfides to sulfoxides catalysed by bacterial cyclohexanone monooxygenases. Chem Commun 20:2303–2307CrossRefGoogle Scholar
  6. 6.
    Hollmann F, Lin PC, Witholt B, Schmid A (2003) Stereospecific biocatalytic epoxidation: the first example of direct regeneration of a FAD-dependent monooxygenase for catalysis. J Am Chem Soc 125(27):8209–8217CrossRefPubMedGoogle Scholar
  7. 7.
    Walsh CT, Chen YCJ (1988) Enzymic Baeyer–Villiger oxidations by flavin-dependent monooxygenases. Angew Chem Int Ed Engl 27(3):333–343CrossRefGoogle Scholar
  8. 8.
    McCormick DB (1970) Flavin derivatives via bromination of the 8-methyl substituent. J Heterocycl Chem 7(2):447–450CrossRefGoogle Scholar
  9. 9.
    Yamamoto S, Katagiri M, Maeno H, Hayaishi O (1965) Salicylate hydroxylase, a monooxygenase requiring flavin adenine dinucleotide I. Purification and general properties. J Biol Chem 240(8):3408–3413PubMedGoogle Scholar
  10. 10.
    Katagiri M, Takemori S, Suzuki K, Yasuda H (1966) Mechanism of the salicylate hydroxylase reaction. J Biol Chem 241(23):5675–5677PubMedGoogle Scholar
  11. 11.
    Uemura T, Kita A, Watanabe Y, Adachi M, Kuroki R, Morimoto Y (2016) The catalytic mechanism of decarboxylative hydroxylation of salicylate hydroxylase revealed by crystal structure analysis at 2.5 Å resolution. Biochem Biophys Res Commun 469(2):158–163CrossRefPubMedGoogle Scholar
  12. 12.
    Schreuder HA, Prick PA, Wierenga RK, Vriend G, Wilson KS, Hol WG, Drenth J (1989) Crystal structure of the p-hydroxybenzoate hydroxylase-substrate complex refined at 1.9 Å resolution: analysis of the enzyme-substrate and enzyme-product complexes. J Mol Biol 208(4):679–696CrossRefPubMedGoogle Scholar
  13. 13.
    Hiromoto T, Fujiwara S, Hosokawa K, Yamaguchi H (2006) Crystal structure of 3-hydroxybenzoate hydroxylase from Comamonas testosteroni has a large tunnel for substrate and oxygen access to the active site. J Mol Biol 364(5):878–896CrossRefPubMedGoogle Scholar
  14. 14.
    Ridder L, Mulholland AJ, Rietjens IM, Vervoort J (1999) Combined quantum mechanical and molecular mechanical reaction pathway calculation for aromatic hydroxylation by p-hydroxybenzoate-3-hydroxylase. J Mol Graph Model 17(3):163–175CrossRefPubMedGoogle Scholar
  15. 15.
    Hicks KA, Yuen ME, Zhen WF, Gerwig TJ, Story RW, Kopp MC, Snider MJ (2016) Structural and biochemical characterization of 6-hydroxynicotinic acid 3-monooxygenase, a novel decarboxylative hydroxylase involved in aerobic nicotinate degradation. Biochemistry 55(24):3432–3446CrossRefPubMedGoogle Scholar
  16. 16.
    Balke K, Schmidt S, Genz M, Bornscheuer UT (2015) Switching the regioselectivity of a cyclohexanone monooxygenase toward (+)-trans-dihydrocarvone by rational protein design. ACS Chem Biol 11(1):38–43CrossRefPubMedGoogle Scholar
  17. 17.
    Li Y, Ding L, Zhang Q, Wang W (2013) MD and QM/MM study on catalytic mechanism of a FAD-dependent enzyme ORF36: for nitro sugar biosynthesis. J Mol Graph Model 44:9–16CrossRefPubMedGoogle Scholar
  18. 18.
    Kobayashi J, Yoshida H, Yagi T, Kamitori S, Hayashi H, Mizutani K, Takahashi N, Mikami B (2017) Role of the Tyr270 residue in 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase from Mesorhizobium loti. J Biosci Bioeng 123(2):154–162CrossRefPubMedGoogle Scholar
  19. 19.
    Asamizu S (2017) Biosynthesis of nitrogen-containing natural products, C7 N aminocyclitols and bis-indoles, from actinomycetes. Biosci Biotechnol Biochem 81(5):871–881CrossRefPubMedGoogle Scholar
  20. 20.
    Ridder L, Harvey JN, Rietjens IM, Vervoort J, Mulholland AJ (2003) Ab initio QM/MM modeling of the hydroxylation step in p-hydroxybenzoate hydroxylase. J Phys Chem B 107(9):2118–2126CrossRefGoogle Scholar
  21. 21.
    Sali A, Blundell TL (1993) Comparative protein modelling by satisfaction of spatial restraints. J Mol Biol 234(3):779–815CrossRefPubMedGoogle Scholar
  22. 22.
    Visitsatthawong S, Chenprakhon P, Chaiyen P, Surawatanawong P (2015) Mechanism of oxygen activation in a flavin-dependent monooxygenase: a nearly barrierless formation of C4a-hydroperoxyflavin via proton-coupled electron transfer. J Am Chem Soc 137(29):9363–9374CrossRefPubMedGoogle Scholar
  23. 23.
    Olsson MH, Søndergaard CR, Rostkowski M, Jensen JH (2011) PROPKA3: consistent treatment of internal and surface residues in empirical pKa predictions. J Chem Theory Comput 7(2):525–537CrossRefPubMedGoogle Scholar
  24. 24.
    Bas DC, Rogers DM, Jensen JH (2008) Very fast prediction and rationalization of pKa values for protein–ligand complexes. Proteins Struct Funct Bioinform 73(3):765–783CrossRefGoogle Scholar
  25. 25.
    Brooks BR, Bruccoleri RE, Olafson BD, States DJ, Swaminathan S, Karplus M (1983) CHARMM: a program for macromolecular energy, minimization, and dynamics calculations. J Comput Chem 4(2):187–217CrossRefGoogle Scholar
  26. 26.
    MacKerel AD Jr, Bashford D, Bellott M Jr, Dunbrack RL, Evanseck JD, Field MJ, Fischer S, Gao J, Guo H, Ha S, Joseph-McCarthy D, Kuchnir L, Kuczera K, Lau FTK, Mattos C, Michnick S, Ngo T, Nguyen DT, Prodhom B, Reiher WE III, Roux B, Schlenkrich M, Smith JC, Stote R, Straub J, Watanabe M, Wiorkiewicz-Kuczera J, Yin D, Karplus M (1998) J Phys Chem B 102:3586–3616CrossRefGoogle Scholar
  27. 27.
    Chen J, Im W, Brooks CL (2006) Balancing solvation and intramolecular interactions: toward a consistent generalized Born force field. J Am Chem Soc 128(11):3728–3736CrossRefPubMedPubMedCentralGoogle Scholar
  28. 28.
    Sherwood P, de Vries AH, Guest MF, Schreckenbach G, Catlow CRA, French SA, Sokol AA, Bromley ST, Thiel W, Billeter S et al (2003) QUASI: a general purpose implementation of the QM/MM approach and its application to problems in catalysis. J Mol Struct THEOCHEM 632(1):1–28CrossRefGoogle Scholar
  29. 29.
    Ahlrichs R, Bär M, Häser M, Horn H, Kölmel C (1989) Electronic structure calculations on workstation computers: the program system turbomole. Chem Phys Lett 162(3):165–169CrossRefGoogle Scholar
  30. 30.
    Smith W, Forester TR (1996) DL_POLY_2. 0: a general-purpose parallel molecular dynamics simulation package. J Mol Graph 14(3):136–141CrossRefPubMedGoogle Scholar
  31. 31.
    Bakowies D, Thiel W (1996) Hybrid models for combined quantum mechanical and molecular mechanical approaches. J Phys Chem 100(25):10580–10594CrossRefGoogle Scholar
  32. 32.
    De Vries AH, Sherwood P, Collins SJ, Rigby AM, Rigutto M, Kramer GJ (1999) Zeolite structure and reactivity by combined quantum-chemical-classical calculations. J Phys Chem B 103(29):6133–6141CrossRefGoogle Scholar
  33. 33.
    Billeter SR, Turner AJ, Thiel W (2000) Linear scaling geometry optimisation and transition state search in hybrid delocalised internal coordinates. Phys Chem Chem Phys 2(10):2177–2186CrossRefGoogle Scholar
  34. 34.
    Becke AD (1993) Density-functional thermochemistry. III. The role of exact exchange. J Chem Phys 98(7):5648–5652CrossRefGoogle Scholar
  35. 35.
    Nocedal J (1980) Updating quasi-Newton matrices with limited storage. Math Comput 35(151):773–782CrossRefGoogle Scholar
  36. 36.
    Liu DC, Nocedal J (1989) On the limited memory BFGS method for large scale optimization. Math Program 45(1):503–528CrossRefGoogle Scholar
  37. 37.
    Banerjee A, Adams N, Simons J, Shepard R (1985) Search for statlonary polnts on surfaces. J Phy Chem 197:52–57CrossRefGoogle Scholar
  38. 38.
    Yu H, Hausinger RP, Tang HZ, Xu P (2014) Mechanism of the 6-hydroxy-3-succinoyl-pyridine 3-monooxygenase flavoprotein from Pseudomonas putida S16. J Biol Chem 289(42):29158–29170CrossRefPubMedGoogle Scholar
  39. 39.
    Wang LH, Tu SC (1984) The kinetic mechanism of salicylate hydroxylase as studied by initial rate measurement, rapid reaction kinetics, and isotope effects. J Biol Chem 259(17):10682–10688PubMedGoogle Scholar
  40. 40.
    Polyak I, Reetz MT, Thiel W (2012) Quantum mechanical/molecular mechanical study on the mechanism of the enzymatic Baeyer–Villiger reaction. J Am Chem Soc 134(5):2732–2741CrossRefPubMedGoogle Scholar
  41. 41.
    Kästner J, Senn HM, Thiel S, Otte N, Thiel W (2006) QM/MM free-energy perturbation compared to thermodynamic integration and umbrella sampling: application to an enzymatic reaction. J Chem Theory Comput 2(2):452–461CrossRefPubMedGoogle Scholar
  42. 42.
    Senn HM, Thiel S, Thiel W (2005) Enzymatic hydroxylation in p-hydroxybenzoate hydroxylase: a case study for QM/MM molecular dynamics. J Chem Theory Comput 1(3):494–505CrossRefPubMedGoogle Scholar
  43. 43.
    Júnior LR, de Oliveira Neto G, Fernandes JR, Kubota LT (2000) Determination of salicylate in blood serum using an amperometric biosensor based on salicylate hydroxylase immobilized in a polypyrrole–glutaraldehyde matrix. Talanta 51(3):547–557CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2018

Authors and Affiliations

  1. 1.School of Chemistry and Chemical EngineeringShandong UniversityJinanChina
  2. 2.Shandong Non-metallic Materials InstituteJinanChina

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