| Structural highlights
Function
CPXB_PRIM2 Functions as a fatty acid monooxygenase (PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:16566047, PubMed:17077084, PubMed:1727637, PubMed:17868686, PubMed:18004886, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028, PubMed:3106359, PubMed:7578081). Catalyzes hydroxylation of fatty acids at omega-1, omega-2 and omega-3 positions (PubMed:1727637, PubMed:21875028). Shows activity toward medium and long-chain fatty acids, with optimum chain lengths of 12, 14 and 16 carbons (lauric, myristic, and palmitic acids). Able to metabolize some of these primary metabolites to secondary and tertiary products (PubMed:1727637). Marginal activity towards short chain lengths of 8-10 carbons (PubMed:1727637, PubMed:18619466). Hydroxylates highly branched fatty acids, which play an essential role in membrane fluidity regulation (PubMed:16566047). Also displays a NADPH-dependent reductase activity in the C-terminal domain, which allows electron transfer from NADPH to the heme iron of the cytochrome P450 N-terminal domain (PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:16566047, PubMed:17077084, PubMed:1727637, PubMed:17868686, PubMed:18004886, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028, PubMed:3106359, PubMed:7578081). Involved in inactivation of quorum sensing signals of other competing bacteria by oxidazing efficiently acyl homoserine lactones (AHLs), molecules involved in quorum sensing signaling pathways, and their lactonolysis products acyl homoserines (AHs) (PubMed:18020460).[1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18]
Publication Abstract from PubMed
P450 dihydroxylase plays a crucial role in steroid drug synthesis by efficiently catalyzing two-step selective hydroxylation reactions. However, natural P450 dihydroxylases are scarce, with poor catalytic performance and intermediate accumulation limiting production. Here, we report a P450 dihydroxylase BM3-H that synthesizes 7alpha,15alpha-diOH-DHEA with 76â¯% selectivity. To enhance 7alpha,15alpha-diOH-DHEA synthesis, we engineered a novel exit channel for the intermediate by modifying key residues in the solvent channel. The triple mutant D182K/E143D/V178A exhibited significant improvements in product concentration (10.08-fold), enzymatic activity (2.16-fold), catalytic efficiency (k(cat)/K(m), 42.32-fold), electron transfer rate (k(ET), 27.14-fold), and coupling efficiency (CE, 3.93-fold). Molecular dynamics simulations revealed that D182K/E143D/V178A created a novel exit channel for 7alpha-OH-DHEA, with channel length, polarity, and steric hindrance influencing enzyme performance. Our approach enhances the overall catalytic performance of P450BM3-H by excavating new intermediate product exit channels, providing theoretical guidance for the design of other enzyme molecules.
Rationally designing P450BM3-H to excavate a novel channel for product exit and enhance overall performance.,Deng Q, Lu ZM, Yuan Z, Feng Y, Zhang L, Shi J, Xu Z, Kofas MAG, Li H Int J Biol Macromol. 2025 Mar 17:142162. doi: 10.1016/j.ijbiomac.2025.142162. PMID:40107536[19]
From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.
References
- ↑ Haines DC, Tomchick DR, Machius M, Peterson JA. Pivotal role of water in the mechanism of P450BM-3. Biochemistry. 2001 Nov 13;40(45):13456-65. PMID:11695892
- ↑ Ost TW, Clark J, Mowat CG, Miles CS, Walkinshaw MD, Reid GA, Chapman SK, Daff S. Oxygen activation and electron transfer in flavocytochrome P450 BM3. J Am Chem Soc. 2003 Dec 10;125(49):15010-20. PMID:14653735 doi:http://dx.doi.org/10.1021/ja035731o
- ↑ Clark JP, Miles CS, Mowat CG, Walkinshaw MD, Reid GA, Daff SN, Chapman SK. The role of Thr268 and Phe393 in cytochrome P450 BM3. J Inorg Biochem. 2006 May;100(5-6):1075-90. Epub 2006 Jan 5. PMID:16403573 doi:10.1016/j.jinorgbio.2005.11.020
- ↑ Budde M, Morr M, Schmid RD, Urlacher VB. Selective hydroxylation of highly branched fatty acids and their derivatives by CYP102A1 from Bacillus megaterium. Chembiochem. 2006 May;7(5):789-94. PMID:16566047 doi:http://dx.doi.org/10.1002/cbic.200500444
- ↑ Girvan HM, Seward HE, Toogood HS, Cheesman MR, Leys D, Munro AW. Structural and spectroscopic characterization of P450 BM3 mutants with unprecedented P450 heme iron ligand sets. New heme ligation states influence conformational equilibria in P450 BM3. J Biol Chem. 2007 Jan 5;282(1):564-72. Epub 2006 Oct 31. PMID:17077084 doi:10.1074/jbc.M607949200
- ↑ Boddupalli SS, Pramanik BC, Slaughter CA, Estabrook RW, Peterson JA. Fatty acid monooxygenation by P450BM-3: product identification and proposed mechanisms for the sequential hydroxylation reactions. Arch Biochem Biophys. 1992 Jan;292(1):20-8. PMID:1727637
- ↑ Huang WC, Westlake AC, Marechal JD, Joyce MG, Moody PC, Roberts GC. Filling a hole in cytochrome P450 BM3 improves substrate binding and catalytic efficiency. J Mol Biol. 2007 Oct 26;373(3):633-51. Epub 2007 Aug 21. PMID:17868686 doi:S0022-2836(07)01086-8
- ↑ Hegde A, Haines DC, Bondlela M, Chen B, Schaffer N, Tomchick DR, Machius M, Nguyen H, Chowdhary PK, Stewart L, Lopez C, Peterson JA. Interactions of substrates at the surface of P450s can greatly enhance substrate potency. Biochemistry. 2007 Dec 11;46(49):14010-7. Epub 2007 Nov 16. PMID:18004886 doi:10.1021/bi701667m
- ↑ Chowdhary PK, Keshavan N, Nguyen HQ, Peterson JA, Gonzalez JE, Haines DC. Bacillus megaterium CYP102A1 oxidation of acyl homoserine lactones and acyl homoserines. Biochemistry. 2007 Dec 18;46(50):14429-37. Epub 2007 Nov 20. PMID:18020460 doi:http://dx.doi.org/10.1021/bi701945j
- ↑ Haines DC, Chen B, Tomchick DR, Bondlela M, Hegde A, Machius M, Peterson JA. Crystal structure of inhibitor-bound P450BM-3 reveals open conformation of substrate access channel. Biochemistry. 2008 Mar 25;47(12):3662-70. Epub 2008 Feb 26. PMID:18298086 doi:10.1021/bi7023964
- ↑ Fasan R, Meharenna YT, Snow CD, Poulos TL, Arnold FH. Evolutionary history of a specialized p450 propane monooxygenase. J Mol Biol. 2008 Nov 28;383(5):1069-80. Epub 2008 Jun 28. PMID:18619466 doi:10.1016/j.jmb.2008.06.060
- ↑ Girvan HM, Toogood HS, Littleford RE, Seward HE, Smith WE, Ekanem IS, Leys D, Cheesman MR, Munro AW. Novel haem co-ordination variants of flavocytochrome P450BM3. Biochem J. 2009 Jan 1;417(1):65-76. PMID:18721129 doi:BJ20081133
- ↑ Whitehouse CJ, Bell SG, Yang W, Yorke JA, Blanford CF, Strong AJ, Morse EJ, Bartlam M, Rao Z, Wong LL. A Highly Active Single-Mutation Variant of P450(BM3) (CYP102A1). Chembiochem. 2009 Jun 2;10(10):1654-1656. PMID:19492389 doi:10.1002/cbic.200900279
- ↑ Girvan HM, Levy CW, Williams P, Fisher K, Cheesman MR, Rigby SE, Leys D, Munro AW. Glutamate-haem ester bond formation is disfavoured in flavocytochrome P450 BM3: characterization of glutamate substitution mutants at the haem site of P450 BM3. Biochem J. 2010 Apr 14;427(3):455-66. PMID:20180779 doi:10.1042/BJ20091603
- ↑ Whitehouse CJ, Yang W, Yorke JA, Rowlatt BC, Strong AJ, Blanford CF, Bell SG, Bartlam M, Wong LL, Rao Z. Structural basis for the properties of two single-site proline mutants of CYP102A1 (P450BM3). Chembiochem. 2010 Dec 10;11(18):2549-56. doi: 10.1002/cbic.201000421. PMID:21110374 doi:http://dx.doi.org/10.1002/cbic.201000421
- ↑ Haines DC, Hegde A, Chen B, Zhao W, Bondlela M, Humphreys JM, Mullin DA, Tomchick DR, Machius M, Peterson JA. A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation. Biochemistry. 2011 Oct 4;50(39):8333-41. Epub 2011 Sep 6. PMID:21875028 doi:10.1021/bi201099j
- ↑ Wen LP, Fulco AJ. 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. 1987 May 15;262(14):6676-82. PMID:3106359
- ↑ Yeom H, Sligar SG, Li H, Poulos TL, Fulco AJ. The role of Thr268 in oxygen activation of cytochrome P450BM-3. Biochemistry. 1995 Nov 14;34(45):14733-40. PMID:7578081
- ↑ Deng Q, Lu ZM, Yuan Z, Feng Y, Zhang L, Shi J, Xu Z, Kofas MAG, Li H. Rationally designing P450BM3-H to excavate a novel channel for product exit and enhance overall performance. Int J Biol Macromol. 2025 Mar 17:142162. PMID:40107536 doi:10.1016/j.ijbiomac.2025.142162
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