Cloning, Expression, Characterization, and Biocatalytic Investigation of the 4-Hydroxyacetophenone Monooxygenase from Pseudomonas putida JD1

ABSTRACT While the number of available recombinant Baeyer-Villiger monooxygenases (BVMOs) has grown significantly over the last few years, there is still the demand for other BVMOs to expand the biocatalytic diversity. Most BVMOs that have been described are dedicated to convert efficiently cyclohexanone and related cyclic aliphatic ketones. To cover a broader range of substrate types and enantio- and/or regioselectivities, new BVMOs have to be discovered. The gene encoding a BVMO identified in Pseudomonas putida JD1 converting aromatic ketones (HAPMO; 4-hydroxyacetophenone monooxygenase) was amplified from genomic DNA using SiteFinding-PCR, cloned, and functionally expressed in Escherichia coli. Furthermore, four other open reading frames could be identified clustered around this HAPMO. It has been suggested that these proteins, including the HAPMO, might be involved in the degradation of 4-hydroxyacetophenone. Substrate specificity studies revealed that a large variety of other arylaliphatic ketones are also converted via Baeyer-Villiger oxidation into the corresponding esters, with preferences for para-substitutions at the aromatic ring. In addition, oxidation of aldehydes and some heteroaromatic compounds was observed. Cycloketones and open-chain ketones were not or poorly accepted, respectively. It was also found that this enzyme oxidizes aromatic ketones such as 3-phenyl-2-butanone with excellent enantioselectivity (E ≫100).

[1]  A. Baeyer,et al.  Einwirkung des Caro'schen Reagens auf Ketone , 1899 .

[2]  G. E. Turfitt.  The microbiological degradation of steroids: 1. The sterol content of soils. , 1943, The Biochemical journal.

[3]  G E Turfitt,et al.  The microbiological degradation of steroids: 4. Fission of the steroid molecule. , 1948, The Biochemical journal.

[4]  A. J. Markovetz,et al.  An enzyme system for aliphatic methyl ketone oxidation. , 1969, Biochemical and biophysical research communications.

[5]  U. K. Laemmli,et al.  Cleavage of Structural Proteins during the Assembly of the Head of Bacteriophage T4 , 1970, Nature.

[6]  P. Trudgill,et al.  The metabolism of cyclohexanol by Acinetobacter NCIB 9871. , 1975, European journal of biochemistry.

[7]  Preparation of methyl ketones by the sequential treatment of carboxylic acids with methyllithium and chlorotrimethylsilane , 1983 .

[8]  J. Sambrook,et al.  Molecular Cloning: A Laboratory Manual , 2001 .

[9]  QUANTITATIVE ANALYSES OF BIOCHEMICAL KINETIC RESOLUTIONS OF ENANTIOMERS , 1983 .

[10]  Allylic and propargylic phenyl selenide oxygenation by cyclohexanone oxygenase: [2,3]-sigmatraopic rearrangement of the enzyme-generated selenoxide , 1986 .

[11]  C. Walsh,et al.  Acinetobacter cyclohexanone monooxygenase: gene cloning and sequence determination , 1988, Journal of bacteriology.

[12]  D. Hartl,et al.  Genetic applications of an inverse polymerase chain reaction. , 1988, Genetics.

[13]  E. Stackebrandt,et al.  Nucleic acid techniques in bacterial systematics , 1991 .

[14]  U. Bornscheuer,et al.  Characterization and enantioselectivity of a recombinant esterase from Pseudomonas fluorescens , 1998 .

[15]  S. Colonna,et al.  Oxidation of organic cyclic sulfites to sulfates: a new reaction catalyzed by cyclohexanone monooxygenase , 1998 .

[16]  First asymmetric oxidation of tertiary amines by cyclohexanone monooxygenase , 1999 .

[17]  F. M. Brown,et al.  Biotransformation of organic sulfides: Part. 10. Formation of chiral ortho- and meta-substituted benzyl methyl sulfoxides by biotransformation using Helminthosporium species NRRL 4671 , 1999 .

[18]  A. Kerridge,et al.  Biotransformation of organic sulfides. Part 11. Preparation of functionalised phenyl propyl sulfoxides using helminthosporium species and Mortierella isabellina , 1999 .

[19]  D. Hopper,et al.  Conversion of 4-Hydroxyacetophenone into 4-Phenyl Acetate by a Flavin Adenine Dinucleotide-Containing Baeyer-Villiger-Type Monooxygenase , 2000, Journal of bacteriology.

[20]  D. Janssen,et al.  4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds. , 2001, European journal of biochemistry.

[21]  J. Woodley,et al.  Large scale production of cyclohexanone monooxygenase from Escherichia coli TOP10 pQR239. , 2001, Enzyme and microbial technology.

[22]  H. Holland.  Biotransformation of organic sulfides. , 2001, Natural product reports.

[23]  P. Lau,et al.  Cloning and Characterization of a Gene Cluster Involved in Cyclopentanol Metabolism in Comamonas sp. Strain NCIMB 9872 and Biotransformations Effected by Escherichia coli-Expressed Cyclopentanone 1,2-Monooxygenase , 2002, Applied and Environmental Microbiology.

[24]  O. White,et al.  Complete genome sequence and comparative analysis of the metabolically versatile Pseudomonas putida KT2440. , 2002, Environmental microbiology.

[25]  P. Kefalas,et al.  A New Protocol for a Regioselective Aldol Condensation as an Alternative Convenient Synthesis of β-Ketols and α,β-Unsaturated Ketones , 2002 .

[26]  M. Mihovilovic,et al.  Monooxygenase‐Mediated Baeyer−Villiger Oxidations , 2002 .

[27]  S. Colonna,et al.  First asymmetric epoxidation catalysed by cyclohexanone monooxygenase , 2002 .

[28]  Marco W. Fraaije,et al.  Baeyer-Villiger monooxygenases, an emerging family of flavin-dependent biocatalysts , 2003 .

[29]  J. Woodley,et al.  Towards large-scale synthetic applications of Baeyer-Villiger monooxygenases. , 2003, Trends in biotechnology.

[30]  D. Janssen,et al.  Substrate Specificity and Enantioselectivity of 4-Hydroxyacetophenone Monooxygenase , 2003, Applied and Environmental Microbiology.

[31]  V. Alphand,et al.  Microbial Transformations, 56. Preparative Scale Asymmetric Baeyer–Villiger Oxidation using a Highly Productive “Two‐in‐One” Resin‐Based in situ SFPR Concept , 2004 .

[32]  Marco W. Fraaije,et al.  Discovery of a thermostable Baeyer–Villiger monooxygenase by genome mining , 2004, Applied Microbiology and Biotechnology.

[33]  Andrea Mattevi,et al.  Crystal structure of a Baeyer-Villiger monooxygenase. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[34]  Guihong Tan,et al.  SiteFinding-PCR: a simple and efficient PCR method for chromosome walking , 2005, Nucleic acids research.

[35]  Anton Berns,et al.  Cancer: Two in one , 2005, Nature.

[36]  V. Alphand,et al.  Microbial transformations 59: first kilogram scale asymmetric microbial Baeyer-Villiger oxidation with optimized productivity using a resin-based in situ SFPR strategy. , 2005, Biotechnology and bioengineering.

[37]  U. Bornscheuer,et al.  Kinetic resolution of 4-hydroxy-2-ketones catalyzed by a Baeyer-Villiger monooxygenase. , 2006, Angewandte Chemie.

[38]  M. Fraaije,et al.  Biocatalytic properties of Baeyer–Villiger monooxygenases in aqueous–organic media , 2006 .

[39]  M. Fraaije,et al.  4-Hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB as an oxidative biocatalyst in the synthesis of optically active sulfoxides , 2006 .

[40]  Jessica Rehdorf,et al.  Enantioselective kinetic resolution of 3-phenyl-2-ketones using Baeyer–Villiger monooxygenases , 2007 .

[41]  U. Bornscheuer,et al.  Cloning, expression, and characterization of a Baeyer–Villiger monooxygenase from Pseudomonas fluorescens DSM 50106 in E. coli , 2007, Applied Microbiology and Biotechnology.

[42]  Jessica Rehdorf,et al.  Cloning, expression and characterization of a Baeyer-Villiger monooxygenase from Pseudomonas putida KT2440 , 2007, Biotechnology Letters.

[43]  M. Gutiérrez,et al.  Preparative scale Baeyer–Villiger biooxidation at high concentration using recombinant Escherichia coli and in situ substrate feeding and product removal process , 2008, Nature Protocols.

[44]  D. Janssen,et al.  Elucidation of the 4-Hydroxyacetophenone Catabolic Pathway in Pseudomonas fluorescens ACB , 2008, Journal of bacteriology.