Characterization of human flavin-containing monooxygenase (FMO) 3 and FMO5 expressed as maltose-binding protein fusions.
The flavin-containing monooxygenase (FMO) family of enzymes oxygenates nucleophilic xenobiotics and endogenous substances. Human FMO3 and FMO5 are the predominant FMO forms in adult liver. These enzymes are naturally membrane-bound, and recombinant proteins are commercially available as microsomal preparations from insect cells (i.e., Supersome FMO). As an alternative, FMO3 has previously been expressed as a soluble protein, through use of an N-terminal maltose-binding protein (MBP) fusion. In the current study, MBP fusions of both human FMO3 and FMO5 were prepared to >90% purity in the presence of detergent and characterized for biochemical and kinetic parameters, and the parameters were compared with those of Supersome FMO samples. Although MBP-FMO enzymes afforded lower rates of turnover than the corresponding Supersome FMOs, both types of FMO showed identical substrate dependencies and similar responses to changes in assay conditions. Of interest, the FMO3 enzymes showed a 2-fold activation of k(cat)/K(m) in the presence of Triton X-100. Oligomeric analysis of MBP-FMO3 also showed disassociation from a high-order oligomeric form to a monomeric status in the presence of Triton X-100. This report serves as the first direct comparison between Supersome FMOs and the corresponding MBP fusions and the first report of a detergent-based activation of k(cat)/K(m) that corresponds to changes in oligomerization.
Reddy
Robert R
RR
Human BioMolecular Research Institute, 5310 Eastgate Mall, San Diego, CA 92121, USA.
Ralph
Erik C
EC
Motika
Meike S
MS
Zhang
Jun
J
Cashman
John R
JR
D006801:Humans; D007700:Kinetics; D058619:Maltose-Binding Proteins / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D010105:Oxygenases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis* / Q000737:chemistry / Q000302:isolation & purification
D058619:Maltose-Binding Proteins; D011993:Recombinant Fusion Proteins; D010105:Oxygenases; C020066:dimethylaniline monooxygenase (N-oxide forming)
Protein Sci. 1999 Aug;8(8):1668-74
10452611
Drug Metab Dispos. 1997 Jul;25(7):790-7
9224773
Biol Pharm Bull. 2001 Nov;24(11):1263-6
11725960
Pharmacogenetics. 2003 Aug;13(8):495-500
12893987
Biochim Biophys Acta. 2005 Jan 17;1703(2):183-9
15680226
Chem Biol Interact. 1995 Apr 28;96(1):57-73
7720105
Chem Res Toxicol. 1990 Jul-Aug;3(4):372-6
2133087
Drug Metab Dispos. 2003 Jul;31(7):854-60
12814961
Biochem Pharmacol. 1998 Oct 15;56(8):1005-12
9776311
Pharmacol Ther. 2005 Jun;106(3):357-87
15922018
J Biochem Mol Toxicol. 2007;21(4):206-15
17721934
Expert Opin Drug Metab Toxicol. 2008 Dec;4(12):1507-21
19040327
Drug Metab Dispos. 1997 Aug;25(8):1001-7
9280409
Biochemistry. 1999 Apr 27;38(17):5588-95
10220347
Arch Biochem Biophys. 1972 May;150(1):116-25
5028071
Chem Biol Interact. 1997 Aug 29;106(1):29-45
9305407
Arch Biochem Biophys. 2010 Jan 1;493(1):26-36
19944667
Arch Biochem Biophys. 2007 Aug 15;464(2):251-9
17531949
Mol Pharmacol. 1990 May;37(5):643-51
2338943
Arch Biochem Biophys. 1984 Aug 15;233(1):50-63
6087744
Gen Pharmacol. 1991;22(3):549-52
1651268
J Biol Chem. 1993 Mar 5;268(7):5048-54
8444881
Arch Biochem Biophys. 2006 Jun 15;450(2):149-56
16620765
Mol Genet Metab. 2009 Jun;97(2):128-35
19321370
Expert Opin Drug Metab Toxicol. 2007 Dec;3(6):831-45
18028028
Methods Mol Biol. 2003;205:99-117
12491882
Drug Metab Dispos. 2006 Jan;34(1):19-26
16183778
J Pharmacol Exp Ther. 1999 Mar;288(3):1251-60
10027866
Methods Mol Biol. 2008;421:169-89
18826055
Drug Metab Dispos. 2008 Sep;36(9):1828-39
18556438