The catalytic copper of Peptidylglycine alpha-Hydroxylating Monooxygenase also plays a critical structural role
Xavier Siebert 1, Betty A. Eipper 2, Richard E. Mains 2, Sean T. Prigge 3, Ninian J. Blackburn 4 and L. Mario Amzel 1*
1 Johns Hopkins School of Medicine
2 University of Connecticut Health Center
3 Johns Hopkins School of Public Health
4 Oregon Health and Science University
* To whom correspondence should be addressed. E-mail: mario{at}neruda.med.jhmi.edu.
Submitted on May 11, 2005
Revised on July 8, 2005
Accepted on 19 July 2005
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Abstract |
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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 Met314 and two His residues as ligands. Mutation of Met314 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 Met314 as a CuM ligand, we determined the structure of the M314I-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 M314I-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 M314I-PHM crystal form and showed that it does not differ from the structure of wt-PHM in the original crystals. M314I-PHM was also shown to be less stable than wt-PHM by Differential Scanning Calorimetry (DSC). Both structural and calorimetric studies point to a structural role for the CuM site, in addition to its established catalytic role.
Key Words:
X-ray crystallography, copper enzymes, electron-transfer, monooxygenase, protein structure
