1yip: Difference between revisions

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Oxidized Peptidylglycine Alpha-Hydroxylating Monooxygenase (PHM) in a New Crystal Form

OverviewOverview

Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.

About this StructureAbout this Structure

1YIP is a Single protein structure of sequence from Rattus norvegicus with as ligand. Active as Peptidylglycine monooxygenase, with EC number 1.14.17.3 Full crystallographic information is available from OCA.

ReferenceReference

The catalytic copper of peptidylglycine alpha-hydroxylating monooxygenase also plays a critical structural role., Siebert X, Eipper BA, Mains RE, Prigge ST, Blackburn NJ, Amzel LM, Biophys J. 2005 Nov;89(5):3312-9. Epub 2005 Aug 12. PMID:16100265

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-[[Image:1yip.gif|left|200px]]<br /><applet load="1yip" size="450" color="white" frame="true" align="right" spinBox="true"
+[[Image:1yip.gif|left|200px]]<br /><applet load="1yip" size="350" color="white" frame="true" align="right" spinBox="true"
 caption="1yip, resolution 2.200&Aring;" />
 '''Oxidized Peptidylglycine Alpha-Hydroxylating Monooxygenase (PHM) in a New Crystal Form'''<br />
 ==Overview==
-Many bioactive peptides require amidation of their carboxy terminus to, exhibit full biological activity. Peptidylglycine alpha-hydroxylating, monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of, the two steps of this reaction, is composed of two domains, each of which, binds one copper atom (CuH and CuM). The CuM site includes Met(314) and, two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized, enzyme, implying that it contributes only marginally to stabilization of, the CuM site. To characterize the role of Met(314) as a CuM ligand, we, determined the structure of the Met(314)Ile-PHM mutant. Since the mutant, protein failed to crystallize in the conditions of the original wild-type, protein, this structure determination required finding a new crystal form., The Met(314)Ile-PHM mutant structure confirms that the mutation does not, abolish CuM binding to the enzyme, but causes other structural, perturbations that affect the overall stability of the enzyme and the, integrity of the CuH site. To eliminate possible effects of crystal, contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM, crystal form and showed that it does not differ from the structure of, wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also, shown to be less stable than wt-PHM by differential scanning calorimetry., Both structural and calorimetric studies point to a structural role for, the CuM site, in addition to its established catalytic role.
+Many bioactive peptides require amidation of their carboxy terminus to exhibit full biological activity. Peptidylglycine alpha-hydroxylating monooxygenase (PHM; EC 1.14.17.3), the enzyme that catalyzes the first of the two steps of this reaction, is composed of two domains, each of which binds one copper atom (CuH and CuM). The CuM site includes Met(314) and two His residues as ligands. Mutation of Met(314) to Ile inactivates PHM, but has only a minimal effect on the EXAFS spectrum of the oxidized enzyme, implying that it contributes only marginally to stabilization of the CuM site. To characterize the role of Met(314) as a CuM ligand, we determined the structure of the Met(314)Ile-PHM mutant. Since the mutant protein failed to crystallize in the conditions of the original wild-type protein, this structure determination required finding a new crystal form. The Met(314)Ile-PHM mutant structure confirms that the mutation does not abolish CuM binding to the enzyme, but causes other structural perturbations that affect the overall stability of the enzyme and the integrity of the CuH site. To eliminate possible effects of crystal contacts, we redetermined the structure of wt-PHM in the Met(314)Ile-PHM crystal form and showed that it does not differ from the structure of wild-type (wt)-PHM in the original crystals. Met(314)Ile-PHM was also shown to be less stable than wt-PHM by differential scanning calorimetry. Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.
 ==About this Structure==
-YIP is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with CU as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Peptidylglycine_monooxygenase Peptidylglycine monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.17.3 1.14.17.3] Full crystallographic information is available from [http://ispc.weizmann.ac.il/oca-bin/ocashort?id=1YIP OCA].
+YIP is a [http://en.wikipedia.org/wiki/Single_protein Single protein] structure of sequence from [http://en.wikipedia.org/wiki/Rattus_norvegicus Rattus norvegicus] with <scene name='pdbligand=CU:'>CU</scene> as [http://en.wikipedia.org/wiki/ligand ligand]. Active as [http://en.wikipedia.org/wiki/Peptidylglycine_monooxygenase Peptidylglycine monooxygenase], with EC number [http://www.brenda-enzymes.info/php/result_flat.php4?ecno=1.14.17.3 1.14.17.3] Full crystallographic information is available from [http://oca.weizmann.ac.il/oca-bin/ocashort?id=1YIP OCA].
 ==Reference==
@@ Line 14: / Line 14: @@
 [[Category: Rattus norvegicus]]
 [[Category: Single protein]]
-[[Category: Amzel, L.M.]]
+[[Category: Amzel, L M.]]
-[[Category: Blackburn, N.J.]]
+[[Category: Blackburn, N J.]]
-[[Category: Eipper, B.A.]]
+[[Category: Eipper, B A.]]
-[[Category: Mains, R.E.]]
+[[Category: Mains, R E.]]
-[[Category: Prigge, S.T.]]
+[[Category: Prigge, S T.]]
 [[Category: Siebert, X.]]
 [[Category: CU]]
@@ Line 27: / Line 27: @@
 [[Category: oxidoreductase]]
-''Page seeded by [http://ispc.weizmann.ac.il/oca OCA ] on Wed Nov 21 06:48:41 2007''
+''Page seeded by [http://oca.weizmann.ac.il/oca OCA ] on Thu Feb 21 16:05:41 2008''