Skip to main content
SpringerLink
Log in

Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1

  • Biotechnologically Relevant Enzymes and Proteins
  • Published:
Applied Microbiology and Biotechnology Aims and scope Submit manuscript

Abstract

CYP102A1 is an efficient medium- to long-chain fatty acid hydroxylase that is able to accept a wide range of non-natural substrates which bear no resemblance to the natural ones. 4-Hexylbenzoic acid (HBA) and 4-nonyloxybenzoic acid (NOBA) were identified as CYP102A1 substrates via screening studies using the BD Oxygen Biosensor System. Spectroscopic binding studies showed that these two substrates bind in the active site of CYP102A1 with K d values of 2.6 ± 0.1 μM for HBA and 1.9 ± 0.2 μM for NOBA. NADPH consumption rates in the presence of HBA and NOBA were 45 ± 1 min−1 and 61 ± 1 min−1, respectively. The coupling efficiency for NADPH was 57% for NOBA, while it was 77% for HBA. During whole-cell biotransformations, HBA was converted into ω−1- and ω−2-hydroxyhexylbenzoic acid, whereas NOBA was oxidized to ω−2-hydroxynonyloxybenzoic acid and ω−2,ω−4-dihydroxynonyloxybenzoic acid. HBA was used as a fatty acid mimic to compare whole-cell biotransformations with cell-free extracts. Whole-cell biotransformations carried out in a biphasic system resulted in 86% conversion of 5 mM HBA, producing 3.8 mM ω−2- and 0.5 mM ω−1-hydroxyhexylbenzoic acid in 4 h with a turnover number of 4.1 min−1, whereas 100% conversion of 5 mM HBA was obtained in 1 h with crude cell extracts and a cofactor regeneration system, giving a turnover number of 10.5 min−1.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5

Similar content being viewed by others

References

  • Barrow SE, Taylor GW (1987) Gas chromatography and mass spectrometry of eicosanoids. In: Benedetto C, McDonald-Gibson RG, Nigam S, Slater TF (eds) Prostaglandins and related compounds: a practical approach series. IRL, Oxford, pp 99–141

    Google Scholar 

  • Boddupalli SS, Estabrook RW, Peterson JA (1990) Fatty acid monooxygenation by cytochrome P-450BM-3. J Biol Chem 265:4233–4239

    CAS  Google Scholar 

  • Boddupalli SS, Pramanik BC, Slaughter CA, Estabrook RW, Peterson JA (1992) Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanism for sequential hydroxylation. Arch Biochem Biophys 292:20–28

    Article  CAS  Google Scholar 

  • Budde M, Morr M, Schmid RD, Urlacher VB (2006) Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium. Chembiochem 7:789–794

    Article  CAS  Google Scholar 

  • Choi S-J, Kim M, Kim SI, Jeon J-K (2003) Microplate assay measurement of cytochrome P450–carbon monoxide complexes. J Biochem Mol Biol 36:332–335

    CAS  Google Scholar 

  • Cryle MJ, Matovic NJ, De Voss JJ (2007) The stereochemistry of fatty acid hydroxylation by cytochrome P450BM3. Tetrahedron Lett 48:133–136

    Article  CAS  Google Scholar 

  • Eiben S (2007) CYP102A P450 monooxygenases: comparative analysis and construction of cytochrome P450 chimeras. Thesis, University of Stuttgart

  • Eiben S, Kaysser L, Maurer S, Kühnel K, Urlacher VB, Schmid RD (2006) Preparative use of isolated CYP102 monooxygenases—a critical appraisal. J Biotechnol 124:662–669

    Article  CAS  Google Scholar 

  • Fujii T, Narikawa T, Sumisa F, Arisawa A, Takeda K, Kato J (2006) Production of α, ω-alkanediols using Escherichia coli expressing a cytochrome P450 from Acinetobacter sp. OC4. Biosci Biotechnol Biochem 70:1379–1385

    Article  CAS  Google Scholar 

  • Graham-Lorence S, Truan G, Peterson JA, Falck JR, Wei S, Helvig C, Capdevila JH (1997) An active site substitution, F87V, converts cytochrome P450 BM-3 into a regio- and stereoselective (14S, 15R)-arachidonic acid epoxygenase. J Biol Chem 272:1127–1135

    Article  CAS  Google Scholar 

  • Girvan HM, Marshall KR, Lawson RJ, Leys D, Joyce MG, Clarkson J, Smith WE, Cheesman MR, Munro AW (2004) Flavocytochrome P450 BM3 mutant A264E undergoes substrate-dependent formation of a novel heme iron ligand set. J Biol Chem 279:23274–23286

    Article  CAS  Google Scholar 

  • Huang W-C, Westlake ACG, Maréchal J-D, Joyce MG, Moody PCE, Roberts GCK (2007) Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency. J Mol Biol 373:633–651

    Article  CAS  Google Scholar 

  • Li Q-S, Ogawa J, Schmid RD, Shimizu S (2001) Residue size at position 87 of cytochrome P450 BM-3 determines its stereoselectivity in propylbenzene and 3-chlorostyrene oxidation. FEBS Lett 508:249–252

    Article  CAS  Google Scholar 

  • Maurer SC, Kühnel K, Kaysser LA, Eiben S, Schmid RD, Urlacher VB (2005) Catalytic hydroxylation in biphasic systems using CYP102A1 mutants. Adv Synth Catal 347:1090–1098

    Article  CAS  Google Scholar 

  • Modi S, Primrose WU, Boyle JMB, Gibson CF, Lian L-Y, Roberts GCK (1995) NMR studies of substrate binding to cytochrome P450 BM3: comparisons to cytochrome P450cam. Biochemistry 34:8982–8988

    Article  CAS  Google Scholar 

  • Mouri T, Michizoe J, Ichinose H, Kamiya N, Goto M (2006) A recombinant Escherichia coli whole cell biocatalyst harbouring a cytochrome P450cam monooxygenase system coupled with enzymatic cofactor regeneration. Appl Microbiol Biotechnol 72:514–520

    Article  CAS  Google Scholar 

  • Narhi LO, Fulco AJ (1982) Phenobarbital induction of a soluble cytochrome P-450-dependent fatty-acid mono-oxygenase in Bacillus megaterium. J Biol Chem 257:2147–2150

    CAS  Google Scholar 

  • Noble MA, Miles CS, Chapman SK, Lysek DA, Mackay AC, Reid GA, Hanzlik RP, Munro AW (1999) Roles of key active-site residues in flavocytochrome P450BM3. Biochem J 339:371–379

    Article  CAS  Google Scholar 

  • Olry A, Schneider-Belhaddad F, Heintz D, Werck-Reichhart D (2007) A medium-throughput screening assay to determine catalytic activities of oxygen-consuming enzymes: a new tool for functional characterization of cytochrome P450 and other oxygenases. Plant J 51:331–340

    Article  CAS  Google Scholar 

  • Omura T, Sato R (1964) Carbon monoxide-binding pigment of liver microsomes. J Biol Chem 239:2370–2378

    CAS  Google Scholar 

  • Ost TW, Miles CS, Murdoch J, Cheung Y, Reid GA, Chapman SK, Munro AW (2000) Rational re-design of the substrate binding site of flavocytochrome P450 BM3. FEBS Lett 486:173–177

    Article  CAS  Google Scholar 

  • Pflug S, Richter SM, Urlacher VB (2007) Development of a fed-batch process for the production of the cytochrome P450 monooxygenase CYP102A1 from Bacillus megaterium in E. coli. J Biotechnol 129:481–488

    Article  CAS  Google Scholar 

  • Ravichandran KG, Boddupalli SS, Hasermann CA, Peterson JA, Deisenhofer J (1993) Crystal structure of hemoprotein domain of P450BM-3, a prototype for microsomal P450’s. Science 261:731–736

    Article  CAS  Google Scholar 

  • Rolo AP, Palmeira MC, Cortopassi AG (2009) Biosensor plates detect mitochondrial physiological regulators and mutations in vivo. Anal Biochem 385:176–178

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning: a laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Schneider S, Wubbolts MG, Sanglard D, Witholt B (1998) Biocatalyst engineering by assembly of fatty acid transport and oxidation activities for in vivo application of cytochrome P-450BM-3 Monooxygenase. Appl Environ Microbiol 64:3784–3790

    CAS  Google Scholar 

  • Schneider S, Wubbolts MG, Oesterhelt G, Sanglard D, Witholt B (1999) Controlled regioselectivity of fatty acid oxidation by whole cells producing cytochrome P450BM-3 monooxygenase under varied dissolved oxygen concentrations. Biotechnol Bioeng 64:333–341

    Article  CAS  Google Scholar 

  • Smit MS, Mokgoro MM, Setati E, Nicaud JM (2005) α, ω-Dicarboxylic acid accumulation by acyl-CoA oxidase deficient mutants of Yarrowia lipolytica. Biotechnol Lett 27:859–864

    Article  CAS  Google Scholar 

  • Sowden RJ, Yasmin S, Rees NH, Bell SG, Wong L-L (2005) Biotransformation of the sesquiterpene (+)-valencene by cytochrome P450cam and P450BM-3. Org Biomol Chem 3:57–64

    Article  CAS  Google Scholar 

  • Tsotsou GE, Cass AEG, Gilardi G (2002) High throughput assay for cytochrome P450 BM3 for screening libraries of substrates and combinatorial mutants. Biosens Bioelectron 17:119–131

    Article  CAS  Google Scholar 

  • Wen LP, Fulco AJ (1987) Cloning of the gene encoding a catalytically self-sufficient cytochrome P-450 fatty acid monooxygenase induced by barbiturates in Bacillus megaterium and its functional expression and regulation in heterologous (Escherichia coli) and homologous (Bacillus megaterium) hosts. J Biol Chem 262:6676–6682

    CAS  Google Scholar 

  • Whitehouse CJC, Bell SG, Tufton HG, Kenny RJP, Ogilvie LCI, Wong L-L (2008a) Evolved CYP102A1 (P450BM3) variants oxidise a range of non-natural substrates and offer new selectivity options. Chem Commun 8:966–968

    Article  Google Scholar 

  • Whitehouse CJC, Bell SG, Wong L-L (2008b) Desaturation of alkylbenzenes by cytochrome P450BM3 (CYP102A1). Chem Eur J 14:10905–10908

    Article  CAS  Google Scholar 

  • Whitehouse CJC, Bell SG, Yang W, Yorke JA, Blanford CF, Strong AJF, Morse EJ, Bartlam M, Rao Z, Wong LL (2009) A highly active single-mutation variant of P450BM3 (CYP102A1). Chembiochem 10:1654–1656

    Article  CAS  Google Scholar 

  • Wodnicka M, Guarino RD, Hemperly JJ, Timmins MR, Stitt D, Pitner JB (2000) Novel fluorescent technology platform for high throughput cytotoxicity and proliferation assays. Biomol Screen 5:141–152

    Article  CAS  Google Scholar 

Download references

Acknowledgments

Financial support by the University of the Free State, the South African National Research Foundation, and the South African Department of Science and Technology/National Research Foundation Centre of Excellence in Catalysis is gratefully acknowledged. We thank Prof. Vlada B. Urlacher for providing the pET28a-CYP102A1 plasmid, as well as Prof. J. Albertyn and Dr. K. Syed for assistance with transformation into E. coli. We also thank Sarel Marais for technical assistance and Charlene Randall for assisting in the preparation of the manuscript.

Author information

Authors and Affiliations

  1. Department of Microbial, Biochemical and Food Biotechnology, University of the Free State, P.O. Box 339, Bloemfontein, 9300, South Africa

    Rama Krishna Gudiminchi & Martha Sophia Smit

Authors
  1. Rama Krishna Gudiminchi
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Martha Sophia Smit
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Martha Sophia Smit.

Additional information

South African DST-NRF Centre of Excellence in Catalysis, c*change

Rights and permissions

Reprints and permissions

About this article

Cite this article

Gudiminchi, R.K., Smit, M.S. Identification and characterization of 4-hexylbenzoic acid and 4-nonyloxybenzoic acid as substrates of CYP102A1. Appl Microbiol Biotechnol 90, 117–126 (2011). https://doi.org/10.1007/s00253-010-3029-x

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00253-010-3029-x

Keywords

Search

Navigation