1ylr: Difference between revisions

Revision as of 17:06, 21 February 2008

The structure of E.coli nitroreductase with bound acetate, crystal form 1

OverviewOverview

The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.

About this StructureAbout this Structure

1YLR is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as 6,7-dihydropteridine reductase, with EC number 1.5.1.34 Full crystallographic information is available from OCA.

ReferenceReference

Structural and mechanistic studies of Escherichia coli nitroreductase with the antibiotic nitrofurazone. Reversed binding orientations in different redox states of the enzyme., Race PR, Lovering AL, Green RM, Ossor A, White SA, Searle PF, Wrighton CJ, Hyde EI, J Biol Chem. 2005 Apr 8;280(14):13256-64. Epub 2005 Jan 31. PMID:15684426

Page seeded by OCA on Thu Feb 21 16:06:40 2008

Proteopedia Page Contributors and Editors (what is this?)Proteopedia Page Contributors and Editors (what is this?)

OCA

@@ Line 1: / Line 1: @@
-[[Image:1ylr.gif|left|200px]]<br /><applet load="1ylr" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1ylr.gif|left|200px]]<br /><applet load="1ylr" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1ylr, resolution 1.70&Aring;" />
 '''The structure of E.coli nitroreductase with bound acetate, crystal form 1'''<br />
 ==Overview==
-The antibiotics nitrofurazone and nitrofurantoin are used in the treatment, of genitourinary infections and as topical antibacterial agents. Their, action is dependent upon activation by bacterial nitroreductase, flavoproteins, including the Escherichia coli nitroreductase (NTR). Here, we show that the products of reduction of these antibiotics by NTR are the, hydroxylamine derivatives. We show that the reduction of nitrosoaromatics, is enzyme-catalyzed, with a specificity constant approximately 10,000-fold, greater than that of the starting nitro compounds. This suggests that the, reduction of nitro groups proceeds through two successive, enzyme-mediated, reactions and explains why the nitroso intermediates are not observed. The, global reaction rate for nitrofurazone determined in this study is over, 10-fold higher than that previously reported, suggesting that the enzyme, is much more active toward nitroaromatics than previously estimated., Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone, complex, nitrofurazone is oriented with its amide group, rather than the, nitro group to be reduced, positioned over the reactive N5 of the FMN, cofactor. Free acetate, which acts as a competitive inhibitor with respect, to NADH, binds in a similar orientation. We infer that the orientation of, bound nitrofurazone depends upon the redox state of the enzyme. We propose, that the charge distribution on the FMN rings, which alters upon, reduction, is an important determinant of substrate binding and reactivity, in flavoproteins with broad substrate specificity.
+The antibiotics nitrofurazone and nitrofurantoin are used in the treatment of genitourinary infections and as topical antibacterial agents. Their action is dependent upon activation by bacterial nitroreductase flavoproteins, including the Escherichia coli nitroreductase (NTR). Here we show that the products of reduction of these antibiotics by NTR are the hydroxylamine derivatives. We show that the reduction of nitrosoaromatics is enzyme-catalyzed, with a specificity constant approximately 10,000-fold greater than that of the starting nitro compounds. This suggests that the reduction of nitro groups proceeds through two successive, enzyme-mediated reactions and explains why the nitroso intermediates are not observed. The global reaction rate for nitrofurazone determined in this study is over 10-fold higher than that previously reported, suggesting that the enzyme is much more active toward nitroaromatics than previously estimated. Surprisingly, in the crystal structure of the oxidized NTR-nitrofurazone complex, nitrofurazone is oriented with its amide group, rather than the nitro group to be reduced, positioned over the reactive N5 of the FMN cofactor. Free acetate, which acts as a competitive inhibitor with respect to NADH, binds in a similar orientation. We infer that the orientation of bound nitrofurazone depends upon the redox state of the enzyme. We propose that the charge distribution on the FMN rings, which alters upon reduction, is an important determinant of substrate binding and reactivity in flavoproteins with broad substrate specificity.
 ==About this Structure==
-YLR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with ACT and FMN as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/6,7-dihydropteridine_reductase 6,7-dihydropteridine reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.34 1.5.1.34] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1YLR OCA].
+YLR is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Escherichia_coli Escherichia coli] with <scene name='pdbligand=ACT:'>ACT</scene> and <scene name='pdbligand=FMN:'>FMN</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/6,7-dihydropteridine_reductase 6,7-dihydropteridine reductase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.1.34 1.5.1.34] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YLR OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Escherichia coli]]
 [[Category: Single protein]]
-[[Category: Green, R.M.]]
+[[Category: Green, R M.]]
-[[Category: Hyde, E.I.]]
+[[Category: Hyde, E I.]]
-[[Category: Lovering, A.L.]]
+[[Category: Lovering, A L.]]
 [[Category: Ossor, A.]]
-[[Category: Race, P.R.]]
+[[Category: Race, P R.]]
-[[Category: Searle, P.F.]]
+[[Category: Searle, P F.]]
-[[Category: White, S.A.]]
+[[Category: White, S A.]]
-[[Category: Wrighton, C.J.]]
+[[Category: Wrighton, C J.]]
 [[Category: ACT]]
 [[Category: FMN]]
@@ Line 27: / Line 27: @@
 [[Category: oxygen-insensitive nad(p)h nitroreductase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 06:53:59 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:06:40 2008''