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==cytochrome P450 BM3h-2G9C6 MRI sensor, no ligand==
==cytochrome P450 BM3h-2G9C6 MRI sensor, no ligand==
<StructureSection load='4dud' size='340' side='right' caption='[[4dud]], [[Resolution|resolution]] 1.85&Aring;' scene=''>
<StructureSection load='4dud' size='340' side='right'caption='[[4dud]], [[Resolution|resolution]] 1.85&Aring;' scene=''>
== Structural highlights ==
== Structural highlights ==
<table><tr><td colspan='2'>[[4dud]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Atcc_14581 Atcc 14581]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4DUD OCA]. For a <b>guided tour on the structure components</b> use [http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4DUD FirstGlance]. <br>
<table><tr><td colspan='2'>[[4dud]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Priestia_megaterium Priestia megaterium]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=4DUD OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=4DUD FirstGlance]. <br>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat"><scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene></td></tr>
</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene></td></tr>
<tr id='related'><td class="sblockLbl"><b>[[Related_structure|Related:]]</b></td><td class="sblockDat">[[4dtw|4dtw]], [[4dty|4dty]], [[4dtz|4dtz]], [[4du2|4du2]], [[4dua|4dua]], [[4dub|4dub]], [[4duc|4duc]], [[4due|4due]], [[4duf|4duf]]</td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[https://proteopedia.org/fgij/fg.htm?mol=4dud FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4dud OCA], [https://pdbe.org/4dud PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=4dud RCSB], [https://www.ebi.ac.uk/pdbsum/4dud PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=4dud ProSAT]</span></td></tr>
<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">CYP102A1, cyp102 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1404 ATCC 14581])</td></tr>
<tr id='activity'><td class="sblockLbl"><b>Activity:</b></td><td class="sblockDat"><span class='plainlinks'>[http://en.wikipedia.org/wiki/Unspecific_monooxygenase Unspecific monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.14.1 1.14.14.1] </span></td></tr>
<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://oca.weizmann.ac.il/oca-docs/fgij/fg.htm?mol=4dud FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=4dud OCA], [http://pdbe.org/4dud PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=4dud RCSB], [http://www.ebi.ac.uk/pdbsum/4dud PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=4dud ProSAT]</span></td></tr>
</table>
</table>
== Function ==
== Function ==
[[http://www.uniprot.org/uniprot/CPXB_BACME CPXB_BACME]] Functions as a fatty acid monooxygenase. Catalyzes hydroxylation of medium and long-chain fatty acids at omega-1, omega-2 and omega-3 positions, with optimum chain lengths of 12-16 carbons (lauric, myristic, and palmitic acids). The reductase domain is required for electron transfer from NADP to cytochrome P450.  
[[https://www.uniprot.org/uniprot/CPXB_PRIM2 CPXB_PRIM2]] Functions as a fatty acid monooxygenase (PubMed:3106359, PubMed:1727637, PubMed:16566047, PubMed:7578081, PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:18004886, PubMed:17077084, PubMed:17868686, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028). 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:3106359, PubMed:1727637, PubMed:16566047, PubMed:7578081, PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:18004886, PubMed:17077084, PubMed:17868686, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028). 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).<ref>PMID:11695892</ref> <ref>PMID:14653735</ref> <ref>PMID:16403573</ref> <ref>PMID:16566047</ref> <ref>PMID:17077084</ref> <ref>PMID:1727637</ref> <ref>PMID:17868686</ref> <ref>PMID:18004886</ref> <ref>PMID:18020460</ref> <ref>PMID:18298086</ref> <ref>PMID:18619466</ref> <ref>PMID:18721129</ref> <ref>PMID:19492389</ref> <ref>PMID:20180779</ref> <ref>PMID:21110374</ref> <ref>PMID:21875028</ref> <ref>PMID:3106359</ref> <ref>PMID:7578081</ref>
<div style="background-color:#fffaf0;">
<div style="background-color:#fffaf0;">
== Publication Abstract from PubMed ==
== Publication Abstract from PubMed ==
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==See Also==
==See Also==
*[[Cytochrome P450|Cytochrome P450]]
*[[Cytochrome P450 3D structures|Cytochrome P450 3D structures]]
== References ==
== References ==
<references/>
<references/>
__TOC__
__TOC__
</StructureSection>
</StructureSection>
[[Category: Atcc 14581]]
[[Category: Large Structures]]
[[Category: Unspecific monooxygenase]]
[[Category: Priestia megaterium]]
[[Category: Arnold, F H]]
[[Category: Arnold FH]]
[[Category: Brustad, E M]]
[[Category: Brustad EM]]
[[Category: Crook, N]]
[[Category: Crook N]]
[[Category: Jasanoff, A]]
[[Category: Jasanoff A]]
[[Category: Lelyveld, V S]]
[[Category: Lelyveld VS]]
[[Category: Martinez, F M]]
[[Category: Martinez FM]]
[[Category: Scholl, T J]]
[[Category: Scholl TJ]]
[[Category: Snow, C D]]
[[Category: Snow CD]]
[[Category: Cytochrome p450]]
[[Category: Directed evolution]]
[[Category: Mri contrast sensor]]
[[Category: Oxidoreductase]]

Revision as of 11:39, 21 September 2022

cytochrome P450 BM3h-2G9C6 MRI sensor, no ligandcytochrome P450 BM3h-2G9C6 MRI sensor, no ligand

Structural highlights

4dud is a 2 chain structure with sequence from Priestia megaterium. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Ligands:
Resources:FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

Function

[CPXB_PRIM2] Functions as a fatty acid monooxygenase (PubMed:3106359, PubMed:1727637, PubMed:16566047, PubMed:7578081, PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:18004886, PubMed:17077084, PubMed:17868686, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028). 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:3106359, PubMed:1727637, PubMed:16566047, PubMed:7578081, PubMed:11695892, PubMed:14653735, PubMed:16403573, PubMed:18004886, PubMed:17077084, PubMed:17868686, PubMed:18298086, PubMed:18619466, PubMed:18721129, PubMed:19492389, PubMed:20180779, PubMed:21110374, PubMed:21875028). 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

New tools that allow dynamic visualization of molecular neural events are important for studying the basis of brain activity and disease. Sensors that permit ligand-sensitive magnetic resonance imaging (MRI) are useful reagents due to the noninvasive nature and good temporal and spatial resolution of MR methods. Paramagnetic metalloproteins can be effective MRI sensors due to the selectivity imparted by the protein active site and the ability to tune protein properties using techniques such as directed evolution. Here, we show that structure-guided directed evolution of the active site of the cytochrome P450-BM3 heme domain produces highly selective MRI probes with submicromolar affinities for small molecules. We report a new, high-affinity dopamine sensor as well as the first MRI reporter for serotonin, with which we demonstrate quantification of neurotransmitter release in vitro. We also present a detailed structural analysis of evolved cytochrome P450-BM3 heme domain lineages to systematically dissect the molecular basis of neurotransmitter binding affinity, selectivity, and enhanced MRI contrast activity in these engineered proteins.

Structure-Guided Directed Evolution of Highly Selective P450-Based Magnetic Resonance Imaging Sensors for Dopamine and Serotonin.,Brustad EM, Lelyveld VS, Snow CD, Crook N, Jung ST, Martinez FM, Scholl TJ, Jasanoff A, Arnold FH J Mol Biol. 2012 May 30. PMID:22659321[19]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.

See Also

References

  1. ↑ 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
  2. ↑ 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
  3. ↑ 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
  4. ↑ 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
  5. ↑ 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
  6. ↑ 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
  7. ↑ 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
  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
  9. ↑ 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
  10. ↑ 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
  11. ↑ 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
  12. ↑ 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
  13. ↑ 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
  14. ↑ 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
  15. ↑ 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
  16. ↑ 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
  17. ↑ 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
  18. ↑ 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
  19. ↑ Brustad EM, Lelyveld VS, Snow CD, Crook N, Jung ST, Martinez FM, Scholl TJ, Jasanoff A, Arnold FH. Structure-Guided Directed Evolution of Highly Selective P450-Based Magnetic Resonance Imaging Sensors for Dopamine and Serotonin. J Mol Biol. 2012 May 30. PMID:22659321 doi:10.1016/j.jmb.2012.05.029

4dud, resolution 1.85Å

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