A Carboxylate Shift Regulates Dioxygen Activation by the Diiron Nonheme β-Hydroxylase CmlA upon Binding of a Substrate-Loaded Nonribosomal Peptide Synthetase.
The first step in the nonribosomal peptide synthetase (NRPS)-based biosynthesis of chloramphenicol is the β-hydroxylation of the precursor l-p-aminophenylalanine (l-PAPA) catalyzed by the monooxygenase CmlA. The active site of CmlA contains a dinuclear iron cluster that is reduced to the diferrous state (WTR) to initiate O2activation. However, rapid O2activation occurs only when WTRis bound to CmlP, the NRPS to which l-PAPA is covalently attached. Here the X-ray crystal structure of WTRis reported, which is very similar to that of the as-isolated diferric enzyme in which the irons are coordinately saturated. X-ray absorption spectroscopy is used to investigate the WTRcluster ligand structure as well as the structures of WTRin complex with a functional CmlP variant (CmlPAT) with and without l-PAPA attached. It is found that formation of the active WTR:CmlPAT-l-PAPA complex converts at least one iron of the cluster from six- to five-coordinate by changing a bidentately bound amino acid carboxylate to monodentate on Fe1. The only bidentate carboxylate in the structure of WTRis E377. The crystal structure of the CmlA variant E377D shows only monodentate carboxylate coordination. Reduced E377D reacts rapidly with O2in the presence or absence of CmlPAT-l-PAPA, showing loss of regulation. However, this variant fails to catalyze hydroxylation, suggesting that E377 has the dual role of coupling regulation of O2reactivity with juxtaposition of the substrate and the reactive oxygen species. The carboxylate shift in response to substrate binding represents a novel regulatory strategy for oxygen activation in diiron oxygenases.
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.|Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455.
Andrew J
AJ
Jasniewski
Department of Biochemistry Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.|Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455.
Cory J
CJ
Knoot
Department of Biochemistry Molecular Biology and Biophysics, University of Minnesota, Minneapolis, Minnesota 55455.|Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455.
John D
JD
Lipscomb
Department of Chemistry, University of Minnesota, Minneapolis, Minnesota 55455.|Center for Metals in Biocatalysis, University of Minnesota, Minneapolis, Minnesota 55455.
Lawrence
L
Que
D002264:Carboxylic Acids / Q000737:chemistry*; D018360:Crystallography, X-Ray; D007501:Iron / Q000737:chemistry*; D007700:Kinetics; D006899:Mixed Function Oxygenases / Q000737:chemistry*; D010100:Oxygen / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry*; D013379:Substrate Specificity; D056928:X-Ray Absorption Spectroscopy
D002264:Carboxylic Acids; D007501:Iron; D006899:Mixed Function Oxygenases; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase; D010100:Oxygen
Biochemistry. 1998 Sep 22;37(38):13359-69
9748343
Inorg Chem. 2005 Mar 7;44(5):1221-9
15732962
J Biol Inorg Chem. 2016 Sep;21(5-6):589-603
27229511
Biochemistry. 2006 Feb 14;45(6):1685-92
16460015
Acta Crystallogr D Biol Crystallogr. 2010 Apr;66(Pt 4):486-501
20383002
Proc Natl Acad Sci U S A. 2010 Aug 31;107(35):15391-6
20713732
Acc Chem Res. 2011 Apr 19;44(4):280-8
21391602
Inorg Chem. 2004 Jul 26;43(15):4579-89
15257585
Biochemistry. 1981 Mar 3;20(5):1210-6
7225324
Structure. 2015 May 5;23(5):882-892
25865244
Chem Biol. 1995 Sep;2(9):409-18
9432288
Structure. 1996 Sep 15;4(9):1053-64
8805591
Biochemistry. 2005 Jun 7;44(22):8138-47
15924433
Chem Rev. 2001 Jun;101(6):1779-808
11709999
Polyhedron. 2013 Jul 13;58:179-189
23878411
EMBO J. 1996 Aug 15;15(16):4081-92
8861937
Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):355-67
21460454
Inorg Chem. 2001 Jul 16;40(15):3656-69
11442362
J Am Chem Soc. 2004 Dec 29;126(51):16692-3
15612685
Acc Chem Res. 2015 Sep 15;48(9):2632-9
26293615
Biochemistry. 2013 Jun 25;52(25):4331-42
23718184
Biotechnology. 1995;28:505-30
8688636
Biochemistry. 2009 Sep 29;48(38):8932-9
19705873
Proc Natl Acad Sci U S A. 2008 May 13;105(19):6858-63
18458342
Biochemistry. 2003 May 20;42(19):5618-31
12741818
J Biol Inorg Chem. 2016 Sep;21(5-6):605-18
27380180
Chem Commun (Camb). 2014 Feb 11;50(12):1408-10
24270942
Biochemistry. 1999 Apr 6;38(14):4423-32
10194363
Inorg Chem. 2005 Nov 28;44(24):8656-8
16296818
Inorg Chem. 2005 Jan 10;44(1):85-99
15627364
J Biol Chem. 1993 Oct 15;268(29):21569-77
8408008
Biochemistry. 2013 Sep 24;52(38):6662-71
23980641
J Mol Biol. 1991 Aug 5;220(3):723-37
1870128
Methods Enzymol. 1997;276:307-26
27754618
Org Biomol Chem. 2007 Jun 7;5(11):1692-4
17520135
Science. 1997 Jan 24;275(5299):515-8
8999792
Biochemistry. 2000 Nov 7;39(44):13503-15
11063587
Eur J Inorg Chem. 2013 Aug;2013(22-23):3858-3866
24587695
Biochemistry. 2005 Feb 1;44(4):1309-15
15667224
Science. 2000 Jan 7;287(5450):122-5
10615044
Acta Crystallogr C. 2005 Dec;61(Pt 12):m529-31
16330839
J Biol Chem. 2015 Oct 16;290(42):25254-72
26324712
Crit Rev Biochem Mol Biol. 2004 Jan-Feb;39(1):21-68
15121720
Acc Chem Res. 2007 Jul;40(7):475-83
17567087
J Am Chem Soc. 2001 Feb 7;123(5):827-38
11456616
Nature. 2015 Feb 19;518(7539):431-4
25607364
Biochemistry. 1999 Nov 16;38(46):15278-86
10563813
J Am Chem Soc. 2008 Jun 4;130(22):7098-109
18479085
Biochemistry. 1998 Oct 20;37(42):14664-71
9778341
J Am Chem Soc. 2011 May 11;133(18):6938-41
21506543
Protein Sci. 1997 Mar;6(3):556-68
9070438
Biochemistry. 2008 Aug 12;47(32):8386-97
18627173
Biochemistry. 2001 Feb 20;40(7):2220-33
11329291
Nature. 1993 Dec 9;366(6455):537-43
8255292
Acc Chem Res. 2004 Jul;37(7):421-9
15260504
J Biol Chem. 1995 Oct 20;270(42):24662-5
7559577
Inorg Chem. 2005 Jun 27;44(13):4546-54
15962961
J Mol Biol. 1991 Apr 5;218(3):583-93
2016748