5jtd

From Proteopedia

(Difference between revisions)

Current revision

Crystal structure of the Ru(bpy)2PhenA functionalized P450 BM3 L407C heme domain mutant in complex with DMSO.

Structural highlights

5jtd is a 2 chain structure with sequence from Priestia megaterium NBRC 15308 = ATCC 14581. Full crystallographic information is available from OCA. For a guided tour on the structure components use FirstGlance.
Method:	X-ray diffraction, Resolution 1.5Å
Ligands:	, ,
Resources:	FirstGlance, OCA, PDBe, RCSB, PDBsum, ProSAT

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

BACKGROUND: In order to perform selective CH functionalization upon visible light irradiation, Ru(II)-diimine functionalized P450 heme enzymes have been developed. The sL407C-1 enzyme containing the Ru(bpy)2PhenA (bpy=2,2'-bipyridine and PhenA=5-acetamido-1,10-phenanthroline) photosensitizer (1) covalently attached to the non-native single cysteine L407C of the P450BM3 heme domain mutant, displays high photocatalytic activity in the selective CH bond hydroxylation of several substrates. METHODS: A combination of X-ray crystallography, site-directed mutagenesis, transient absorption measurements and enzymatic assays was used to gain insights into its photocatalytic activity and electron transfer pathway. RESULTS: The crystal structure of the sL407C-1 enzyme was solved in the open and closed conformations revealing a through-space electron transfer pathway involving highly conserved, F393 and Q403, residues. Several mutations of these residues (F393A, F393W or Q403W) were introduced to probe their roles in the overall reaction. Transient absorption measurements confirm rapid electron transfer as heme reduction is observed in all four hybrid enzymes. Compared to the parent sL407C-1, photocatalytic activity was negligible in the dF393A-1 enzyme while 60% increase in activity with total turnover numbers of 420 and 90% product conversion was observed with the dQ403W-1 mutant. CONCLUSIONS: In the sL407C-1 enzyme, the photosensitizer is ideally located to rapidly deliver electrons, using the naturally occurring electron transfer pathway, to the heme center in order to activate molecular dioxygen and sustain photocatalytic activity. GENERAL SIGNIFICANCE: The results shed light on the design of efficient light-driven biocatalysts and the approach can be generalized to other members of the P450 superfamily.

Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies.,Spradlin J, Lee D, Mahadevan S, Mahomed M, Tang L, Lam Q, Colbert A, Shafaat OS, Goodin D, Kloos M, Kato M, Cheruzel LE Biochim Biophys Acta. 2016 Sep 14;1864(12):1732-1738. doi:, 10.1016/j.bbapap.2016.09.005. PMID:27639964^[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
↑ Spradlin J, Lee D, Mahadevan S, Mahomed M, Tang L, Lam Q, Colbert A, Shafaat OS, Goodin D, Kloos M, Kato M, Cheruzel LE. Insights into an efficient light-driven hybrid P450 BM3 enzyme from crystallographic, spectroscopic and biochemical studies. Biochim Biophys Acta. 2016 Sep 14;1864(12):1732-1738. doi:, 10.1016/j.bbapap.2016.09.005. PMID:27639964 doi:http://dx.doi.org/10.1016/j.bbapap.2016.09.005

1 Structural highlights
2 Function
3 Publication Abstract from PubMed
4 See Also
5 References

Proteopedia Page Contributors and Editors (what is this?)

OCA

Retrieved from "http://52.214.119.220/wiki/index.php/5jtd"

Categories: Large Structures | Priestia megaterium NBRC 15308 = ATCC 14581 | Kloos M

@@ Line 3: / Line 3: @@
 <StructureSection load='5jtd' size='340' side='right'caption='[[5jtd]], [[Resolution|resolution]] 1.50&Aring;' scene=''>
 == Structural highlights ==
-<table><tr><td colspan='2'>[[5jtd]] is a 2 chain structure with sequence from [http://en.wikipedia.org/wiki/Bacmb Bacmb]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5JTD OCA]. For a <b>guided tour on the structure components</b> use [http://proteopedia.org/fgij/fg.htm?mol=5JTD FirstGlance]. <br>
+<table><tr><td colspan='2'>[[5jtd]] is a 2 chain structure with sequence from [https://en.wikipedia.org/wiki/Priestia_megaterium_NBRC_15308_=_ATCC_14581 Priestia megaterium NBRC 15308 = ATCC 14581]. Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=5JTD OCA]. For a <b>guided tour on the structure components</b> use [https://proteopedia.org/fgij/fg.htm?mol=5JTD FirstGlance]. <br>
-</td></tr><tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=DMS:DIMETHYL+SULFOXIDE'>DMS</scene>, <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=RU8:BIS(2,2-BIPYRIDINE-KAPPA~2~N~1~,N~1~)[2-IODO-N-(1,10-PHENANTHROLIN-5-YL-KAPPA~2~N~1~,N~10~)ACETAMIDE]RUTHENIUM(2+)'>RU8</scene></td></tr>
+</td></tr><tr id='method'><td class="sblockLbl"><b>[[Empirical_models|Method:]]</b></td><td class="sblockDat" id="methodDat">X-ray diffraction, [[Resolution|Resolution]] 1.5&#8491;</td></tr>
-<tr id='gene'><td class="sblockLbl"><b>[[Gene|Gene:]]</b></td><td class="sblockDat">cyp102A1, cyp102, BG04_163 ([http://www.ncbi.nlm.nih.gov/Taxonomy/Browser/wwwtax.cgi?mode=Info&srchmode=5&id=1348623 BACMB])</td></tr>
+<tr id='ligand'><td class="sblockLbl"><b>[[Ligand|Ligands:]]</b></td><td class="sblockDat" id="ligandDat"><scene name='pdbligand=DMS:DIMETHYL+SULFOXIDE'>DMS</scene>, <scene name='pdbligand=HEM:PROTOPORPHYRIN+IX+CONTAINING+FE'>HEM</scene>, <scene name='pdbligand=RU8:BIS(2,2-BIPYRIDINE-KAPPA~2~N~1~,N~1~)[2-IODO-N-(1,10-PHENANTHROLIN-5-YL-KAPPA~2~N~1~,N~10~)ACETAMIDE]RUTHENIUM(2+)'>RU8</scene></td></tr>
-<tr id='resources'><td class="sblockLbl"><b>Resources:</b></td><td class="sblockDat"><span class='plainlinks'>[http://proteopedia.org/fgij/fg.htm?mol=5jtd FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5jtd OCA], [http://pdbe.org/5jtd PDBe], [http://www.rcsb.org/pdb/explore.do?structureId=5jtd RCSB], [http://www.ebi.ac.uk/pdbsum/5jtd PDBsum], [http://prosat.h-its.org/prosat/prosatexe?pdbcode=5jtd ProSAT]</span></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=5jtd FirstGlance], [http://oca.weizmann.ac.il/oca-bin/ocaids?id=5jtd OCA], [https://pdbe.org/5jtd PDBe], [https://www.rcsb.org/pdb/explore.do?structureId=5jtd RCSB], [https://www.ebi.ac.uk/pdbsum/5jtd PDBsum], [https://prosat.h-its.org/prosat/prosatexe?pdbcode=5jtd ProSAT]</span></td></tr>
 </table>
 == Function ==
-[[http://www.uniprot.org/uniprot/CPXB_BACMB CPXB_BACMB]] 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>
+[https://www.uniprot.org/uniprot/CPXB_PRIM2 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).<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;">
 == Publication Abstract from PubMed ==
@@ Line 26: / Line 26: @@
 __TOC__
 </StructureSection>
-[[Category: Bacmb]]
 [[Category: Large Structures]]
-[[Category: Kloos, M]]
+[[Category: Priestia megaterium NBRC 15308 = ATCC 14581]]
-[[Category: Electron transfer]]
+[[Category: Kloos M]]
-[[Category: Hybrid p450 bm3 enzyme]]
-[[Category: Oxidoreductase]]
-[[Category: Photocatalytic activity]]
-[[Category: Photosensitizer]]

5jtd

From Proteopedia

Current revision

Crystal structure of the Ru(bpy)2PhenA functionalized P450 BM3 L407C heme domain mutant in complex with DMSO.

Structural highlights

Function

Publication Abstract from PubMed

See Also

References

Contents

Proteopedia Page Contributors and Editors (what is this?)

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