1tiw: Difference between revisions

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Crystal structure of E. coli PutA proline dehydrogenase domain (residues 86-669) complexed with L-Tetrahydro-2-furoic acid

OverviewOverview

Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.

About this StructureAbout this Structure

1TIW is a Single protein structure of sequence from Escherichia coli with and as ligands. Active as Proline dehydrogenase, with EC number 1.5.99.8 Full crystallographic information is available from OCA.

ReferenceReference

Structures of the Escherichia coli PutA proline dehydrogenase domain in complex with competitive inhibitors., Zhang M, White TA, Schuermann JP, Baban BA, Becker DF, Tanner JJ, Biochemistry. 2004 Oct 5;43(39):12539-48. PMID:15449943

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-[[Image:1tiw.jpg|left|200px]]<br /><applet load="1tiw" size="450" color="white" frame="true" align="right" spinBox="true"
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 caption="1tiw, resolution 2.00&Aring;" />
 '''Crystal structure of E. coli PutA proline dehydrogenase domain (residues 86-669) complexed with L-Tetrahydro-2-furoic acid'''<br />
 ==Overview==
-Proline dehydrogenase (PRODH) catalyzes the first step of proline, catabolism, the flavin-dependent oxidation of proline to, Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study, of the PRODH active site of the multifunctional Escherichia coli proline, utilization A (PutA) protein using X-ray crystallography, enzyme kinetic, measurements, and site-directed mutagenesis. Structures of the PutA PRODH, domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) =, 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The, discovery of acetate as a competitive inhibitor suggests that the carboxyl, is the minimum functional group recognized by the active site, and the, structures show how the enzyme exploits hydrogen-bonding and nonpolar, interactions to optimize affinity for the substrate. The PRODH/L-THFA, complex is the first structure of PRODH with a five-membered ring proline, analogue bound in the active site and thus provides new insights into, substrate recognition and the catalytic mechanism. The ring of L-THFA is, nearly parallel to the middle ring of the FAD isoalloxazine, with the, inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct, hydride transfer as a plausible mechanism. Mutation of conserved active, site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe, loss in thermostability. These changes are consistent with the location of, Leu432 in the hydrophobic core near residues that directly contact FAD., Our results suggest that the molecular basis for increased plasma proline, levels in schizophrenic subjects carrying the missense mutation L441P is, due to decreased stability of human PRODH2.
+Proline dehydrogenase (PRODH) catalyzes the first step of proline catabolism, the flavin-dependent oxidation of proline to Delta(1)-pyrroline-5-carboxylate. Here we present a structure-based study of the PRODH active site of the multifunctional Escherichia coli proline utilization A (PutA) protein using X-ray crystallography, enzyme kinetic measurements, and site-directed mutagenesis. Structures of the PutA PRODH domain complexed with competitive inhibitors acetate (K(i) = 30 mM), L-lactate (K(i) = 1 mM), and L-tetrahydro-2-furoic acid (L-THFA, K(i) = 0.2 mM) have been determined to high-resolution limits of 2.1-2.0 A. The discovery of acetate as a competitive inhibitor suggests that the carboxyl is the minimum functional group recognized by the active site, and the structures show how the enzyme exploits hydrogen-bonding and nonpolar interactions to optimize affinity for the substrate. The PRODH/L-THFA complex is the first structure of PRODH with a five-membered ring proline analogue bound in the active site and thus provides new insights into substrate recognition and the catalytic mechanism. The ring of L-THFA is nearly parallel to the middle ring of the FAD isoalloxazine, with the inhibitor C5 atom 3.3 A from the FAD N5. This geometry suggests direct hydride transfer as a plausible mechanism. Mutation of conserved active site residue Leu432 to Pro caused a 5-fold decrease in k(cat) and a severe loss in thermostability. These changes are consistent with the location of Leu432 in the hydrophobic core near residues that directly contact FAD. Our results suggest that the molecular basis for increased plasma proline levels in schizophrenic subjects carrying the missense mutation L441P is due to decreased stability of human PRODH2.
 ==About this Structure==
-TIW 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 FAD and TFB as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Proline_dehydrogenase Proline dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.99.8 1.5.99.8] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1TIW OCA].
+TIW 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=FAD:'>FAD</scene> and <scene name='pdbligand=TFB:'>TFB</scene> as [http://en.wikipedia.org/wiki/ligands ligands]. Active as [http://en.wikipedia.org/wiki/Proline_dehydrogenase Proline dehydrogenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.5.99.8 1.5.99.8] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1TIW OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Proline dehydrogenase]]
 [[Category: Single protein]]
-[[Category: Baban, B.A.]]
+[[Category: Baban, B A.]]
-[[Category: Becker, D.F.]]
+[[Category: Becker, D F.]]
-[[Category: Schuermann, J.P.]]
+[[Category: Schuermann, J P.]]
-[[Category: Tanner, J.J.]]
+[[Category: Tanner, J J.]]
-[[Category: White, T.A.]]
+[[Category: White, T A.]]
 [[Category: Zhang, M.]]
 [[Category: FAD]]
@@ Line 27: / Line 27: @@
 [[Category: proline catabolism]]
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