4-hydroxyacetophenone monooxygenase - Pseudomonas fluorescens

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Reviewed, UniProtKB/Swiss-Prot Q93TJ5 (HAPMO_PSEFL)

Last modified May 5, 2009. Version 44. History...

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Names and origin · Protein attributes · General annotation (Comments) · Ontologies · Sequence annotation (Features) · Sequences · References · Cross-references · Entry information · Relevant documents

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Names and origin

Protein names

Recommended name:
    4-hydroxyacetophenone monooxygenase
      Short name=HAPMO
    EC=1.14.13.84
Alternative name(s):
    Baeyer-Villiger monooxygenase
      Short name=BVMO

Gene names

Name:

hapE

Organism

Pseudomonas fluorescens

Taxonomic identifier

294 [NCBI]

Taxonomic lineage

Bacteria › Proteobacteria › Gammaproteobacteria › Pseudomonadales › Pseudomonadaceae › Pseudomonas

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Protein attributes

Sequence length	640 AA.
Sequence status	Complete.
Sequence processing	The displayed sequence is further processed into a mature form.
Protein existence	Evidence at protein level.

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General annotation (Comments)

Function	Catalyzes a Baeyer-Villiger oxidation reaction, i.e., the insertion of an oxygen atom into a carbon-carbon bond adjacent to a carbonyl, which converts ketones to esters. Can oxidize a wide range of acetophenone derivatives. Highest activity occurs with compounds bearing an electron-donating substituent at the para position of the aromatic ring, e.g. 4-hydroxyacetophenone and 4-aminoacetophenone, leading to the formation of 4-hydroxyphenyl acetate and 4-aminophenyl acetate, respectively. Is also able to oxidize sulfides. Ref.1 Ref.3
Catalytic activity	(4-hydroxyphenyl)ethan-1-one + NADPH + O₂ = 4-hydroxyphenyl acetate + NADP⁺ + H₂O.
Cofactor	Binds 1 FAD per subunit. Ref.1
Enzyme regulation	Inhibited by amino-NADP⁺. Ref.4
Subunit structure	Homodimer. Ref.1
Sequence similarities	Belongs to the FAD-binding monooxygenase family.
Biophysicochemical properties	Kinetic parameters: K_M=12 µM for NADPH (at pH 7.5) Ref.4 K_M=1.44 mM for NADH (at pH 7.5) K_M=9.2 µM for 4-hydroxyacetophenone (at pH 7.5) K_M=2.4 µM for 4-hydroxypropiophenone (at pH 7.5) K_M=101 µM for 4-hydroxybenzaldehyde (at pH 8.0) K_M=0.82 µM for 4-aminoacetophenone (at pH 7.5) K_M=1.04 mM for 4-fluoroacetophenone (at pH 8.0) K_M=161 µM for 4-methylacetophenone (at pH 8.0) K_M=541 µM for 4-methoxyacetophenone (at pH 8.0) K_M=610 µM for 2-hydroxyacetophenone (at pH 7.5) K_M=1.4 mM for 3-hydroxyacetophenone (at pH 7.5) K_M=1.6 mM for benzaldehyde (at pH 7.5) K_M=2.27 mM for acetophenone (at pH 8.0) K_M=530 µM for propiophenone (at pH 7.5) K_M=2 mM for butyrophenone (at pH 7.5) K_M=540 µM for isobutyrophenone (at pH 7.5) K_M=1.4 mM for methylphenyl sulfide (at pH 7.5) K_M=370 µM for methyl 4-tolyl sulfide (at pH 7.5) K_M=1.2 mM for 2-acetylpyridine (at pH 7.5) K_M=1.9 mM for 4-acetylpyridine (at pH 7.5) K_M=330 µM for 2-acetylpyrrole (at pH 7.5) K_M=410 µM for 2-pyrrole carboxaldehyde (at pH 7.5) K_M=4.8 mM for acetylcyclohexane (at pH 7.5) K_M=3 mM for cyclohexane carboxaldehyde (at pH 7.5) K_M=29 mM for hydroxyacetone (at pH 7.5) K_M=23 mM for 3-chloro-2-butanone (at pH 7.5) K_M=4.9 mM for 2,4-pentanedione (at pH 7.5) K_M=3.3 mM for rac-bicyclo[3.2.0]hept-2-en-6-one (at pH 7.5) K_M=380 µM for 4-hydroxy-3-methylacetophenone (at pH 8.0) pH dependence: Optimum pH is 7.5. Temperature dependence: Optimum temperature is 30 degrees Celsius.
Mass spectrometry	Molecular mass is 77610±10 Da from positions 2 - 640. Determined by ESI. The measured mass is that of a monomer in complex with FAD cofactor. Ref.5

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Ontologies

Keywords
Ligand	FAD Flavoprotein NADP
Molecular function	Monooxygenase Oxidoreductase
Technical term	Direct protein sequencing
Gene Ontology (GO)
Biological process	oxidation reduction Inferred from electronic annotation. Source: UniProtKB-KW
Molecular function	4-hydroxyacetophenone monooxygenase activity Inferred from electronic annotation. Source: EC FAD binding Inferred from electronic annotation. Source: InterPro NADP or NADPH binding Inferred from electronic annotation. Source: InterPro flavin-containing monooxygenase activity Inferred from electronic annotation. Source: InterPro
Complete GO annotation...

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Sequence annotation (Features)

Feature key

Position(s)

Length

Description

Graphical view

Feature identifier

Molecule processing

Initiator methionine

Removed Ref.1

Chain

2 – 640

639

4-hydroxyacetophenone monooxygenase

PRO_0000287886

Regions

Nucleotide binding

191 – 192

FAD By similarity

Sites

Binding site

152

FAD By similarity

Binding site

171

FAD By similarity

Binding site

180

FAD By similarity

Binding site

197

FAD By similarity

Binding site

239

FAD; via amide nitrogen and carbonyl oxygen By similarity

Binding site

275

FAD By similarity

Site

440

Transition state stabilizer Potential

Experimental info

Mutagenesis

296

H → A: Almost no activity. Ref.2

Mutagenesis

300

W → A or Y: Impared folding. Ref.2

Mutagenesis

339

R → A: Largely decreased affinity for NADPH. Ref.4

Mutagenesis

439

K → A or F: 100-fold decrease in catalytic efficiency with NADPH. 4-fold higher efficiency with NADH. Ref.4

Mutagenesis

439

K → N: 20-fold decrease in catalytic efficiency with NADPH. 6-fold higher efficiency with NADH. Ref.4

Mutagenesis

439

K → P: 20-fold decrease in catalytic activity. Ref.4

Mutagenesis

440

R → A: No activity. Retains high affinity toward NADPH. Ref.4

Mutagenesis

490

G → A: Retains activity, but shows impaired binding properties of NADPH. Ref.1

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Sequences

Sequence

Length

Mass (Da)

Tools

Q93TJ5-1 [UniParc].

Last modified December 1, 2001. Version 1.
Checksum: 60750A318723DA6A
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FASTA

640

71,957

        10         20         30         40         50         60 
MSAFNTTLPS LDYDDDTLRE HLQGADIPTL LLTVAHLTGD LQILKPNWKP SIAMGVARSG 

        70         80         90        100        110        120 
MDLETEAQVR EFCLQRLIDF RDSGQPAPGR PTSDQLHILG TWLMGPVIEP YLPLIAEEAV 

       130        140        150        160        170        180 
TAEEDLRAPR WHKDHVASGR DFKVVIIGAG ESGMIAALRF KQAGVPFVIY EKGNDVGGTW 

       190        200        210        220        230        240 
RENTYPGCRV DINSFWYSFS FARGIWDDCF APAPQVFAYM QAVAREHGLY EHIRFNTEVS 

       250        260        270        280        290        300 
DAHWDESTQR WQLLYRDSEG QTQVDSNVVV FAVGQLNRPM IPAIPGIETF KGPMFHSAQW 

       310        320        330        340        350        360 
DHDVDWSGKR VGVIGTGASA TQFIPQLAQT AAELKVFART TNWLLPTPDL HEKISDSCKW 

       370        380        390        400        410        420 
LLAHVPHYSL WYRVAMAMPQ SVGFLEDVMV DVGYPPTELA VSARNDRLRQ DISAWMEPQF 

       430        440        450        460        470        480 
ADRPDLREVL IPDSPVGGKR IVRDNGTWIS TLKRDNVSMI RQPIEVITPK GICCVDGTEH 

       490        500        510        520        530        540 
EFDLIVYGTG FHASKFLMPI NVTGRDGVAL HDVWKGDDAR AYLGMTVPQF PNMFCMYGPN 

       550        560        570        580        590        600 
TGLVVYSTVI QFSEMTASYI VDAVRLLLEG GHQSMEVKTP VFESYNQRVD EGNALRAWGF 

       610        620        630        640 
SKVNSWYKNS KGRVTQNFPF TAVEFWQRTH SVEPTDYQLG

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References

[1]	"4-hydroxyacetophenone monooxygenase from Pseudomonas fluorescens ACB. A novel flavoprotein catalyzing Baeyer-Villiger oxidation of aromatic compounds." Kamerbeek N.M., Moonen M.J.H., van der Ven J.G.M., van Berkel W.J.H., Fraaije M.W., Janssen D.B. Eur. J. Biochem. 268:2547-2557(2001) [PubMed: 11322873] [Abstract] Cited for: NUCLEOTIDE SEQUENCE [GENOMIC DNA], PROTEIN SEQUENCE OF 2-21, FUNCTION, MUTAGENESIS OF GLY-490, BIOPHYSICOCHEMICAL PROPERTIES, COFACTOR, SUBUNIT. Strain: ACB.
[2]	"Identification of a Baeyer-Villiger monooxygenase sequence motif." Fraaije M.W., Kamerbeek N.M., van Berkel W.J.H., Janssen D.B. FEBS Lett. 518:43-47(2002) [PubMed: 11997015] [Abstract] Cited for: MUTAGENESIS OF HIS-296 AND TRP-300. Strain: ACB.
[3]	"Substrate specificity and enantioselectivity of 4-hydroxyacetophenone monooxygenase." Kamerbeek N.M., Olsthoorn A.J.J., Fraaije M.W., Janssen D.B. Appl. Environ. Microbiol. 69:419-426(2003) [PubMed: 12514023] [Abstract] Cited for: FUNCTION, SUBSTRATE SPECIFICITY, BIOPHYSICOCHEMICAL PROPERTIES. Strain: ACB.
[4]	"Identifying determinants of NADPH specificity in Baeyer-Villiger monooxygenases." Kamerbeek N.M., Fraaije M.W., Janssen D.B. Eur. J. Biochem. 271:2107-2116(2004) [PubMed: 15153101] [Abstract] Cited for: MUTAGENESIS OF ARG-339; LYS-439 AND ARG-440, KINETIC PARAMETERS, ENZYME REGULATION. Strain: ACB.
[5]	"Coenzyme binding during catalysis is beneficial for the stability of 4-hydroxyacetophenone monooxygenase." van den Heuvel R.H.H., Tahallah N., Kamerbeek N.M., Fraaije M.W., van Berkel W.J.H., Janssen D.B., Heck A.J.R. J. Biol. Chem. 280:32115-32121(2005) [PubMed: 16049018] [Abstract] Cited for: MASS SPECTROMETRY, REACTION MECHANISM.

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Cross-references

Sequence databases
	AF355751 Genomic DNA. Translation: AAK54073.1.
3D structure databases
ModBase	Search...
Enzyme and pathway databases
BRENDA	1.14.13.84. 329.
Family and domain databases
InterPro	IPR013027. FAD_pyr_nucl-diS_OxRdtase. IPR000960. Flavin_mOase. [Graphical view]
Pfam	PF00743. FMO-like. 1 hit. [Graphical view]
PRINTS	PR00368. FADPNR.
ProDom	PD000139. FAD_pyr_redox. 1 hit. [Graphical view] [Entries sharing at least one domain]
ProtoNet	Search...

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Entry information

Entry name

HAPMO_PSEFL

Accession

Primary (citable) accession number: Q93TJ5

Entry history

Integrated into UniProtKB/Swiss-Prot:	May 15, 2007
Last sequence update:	December 1, 2001
Last modified:	May 5, 2009
This is version 44 of the entry and version 1 of the sequence. [Complete history]

Entry status

Reviewed (UniProtKB/Swiss-Prot)

Annotation project

HAMAP (High-quality Automated and Manual Annotation of microbial Proteomes)

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Relevant documents

SIMILARITY comments

Index of protein domains and families

Names and origin

Protein attributes

General annotation (Comments)

Ontologies

Sequence annotation (Features)

Molecule processing

Regions

Sites

Experimental info

Sequences

References

Cross-references

Sequence databases

3D structure databases

Enzyme and pathway databases

Family and domain databases

Entry information

Relevant documents