Project #2: Peptide Deformylase

 

*    Background

*    Systems of Interest

*    Research Goals and Projects

*    Reading

 (back to main research page)

 

 

Background:

Peptide deformylase (PDF) catalyzes the hydrolytic cleavage of the formyl group at the N-terminus of nascent eubacterial proteins during protein synthesis.  As PDF is essential for bacterial survival but absent in higher animals, PDF constitutes a promising target for a new class of antibacterial agents, making investigation of the structure and function of this enzyme an important endeavor.  PDF is also of great interest from the bioinorganic viewpoint, as it is the only example of an iron metalloamidase.  Other metallopeptidases use ZnII as the metal ion and the structural and functional characteristics of PDF would otherwise indicate that the enzyme belongs in the mononuclear ZnII enzyme family.  The choice of iron by nature is intriguing considering the inherent instability of FeII towards oxidation and that PDF catalyzes a non-redox-active reaction.

Crystal structures of the full-length protein indicate the metal ligands to be Cys90, His132, and His136 in the HEXXH motif and a water molecule or hydroxide ion.  The presence of the cysteinate sulfur ligand as part of the mixed N,S environment is rather uncommon; an N or O donor from His or Asp/Glu is more typical in “zinc” metallopeptidases.  A network of hydrogen bonds aligns the metal-coordinating ligands, and substitution of FeII by ZnII or NiII leads to only minimal structural changes.  High-resolution crystal structures of PDF in complex with formate show differences in formate binding modes when FeII, CoII, and ZnII are present.

The proposed deformylation mechanism (Figure 1) begins with nucleophilic attack of the hydroxide ligand on the carbonyl carbon of the N-terminal formyl group of the peptide.  This is followed by the transition from a tetrahedral to a five-coordinate metal center and subsequent formation of an enzyme-formate complex.  Hydrogen bonds from spatially close residues stabilize charge moieties throughout the mechanism.  The metal functions as an electron-withdrawing group to favor deprotonation of the metal-bound water to yield a hydroxide ligand and as a Lewis acid to activate the bound carbonyl substrate, the latter function of which is likely affected by the presence of the cysteinate ligand.  Interestingly, catalytic activity is lost in ZnII-substituted forms, but maintained with NiII and CoII.  Tighter binding of the ZnII ion is proposed to lead to the inability of zinc to change between tetrahedral and five-fold coordination during catalysis.

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(back to top)

 

Systems of Interest:

 

 

Highly promising biomimetic models for PDF have recently been developed by Goldberg and co-workers.  An N2Sthiolate ligand (Figure 2), 2-methyl-1-[methyl-(2-pyridin-2-yl-ethyl)amino]propane-2-thiol (or PATH, pyridine-amine-thiolate system), has been demonstrated to bind ZnII and CoII, bears an aliphatic thiolate as a mimic of the cysteine donor in PDF, and yields monomeric metal complexes.  The hydrolysis of 4-nitrophenyl acetate by the (PATH)ZnII-hydroxide complex has also been observed.  Another possible biomimetic system is a series of pseudotetrahedral Zn(II) complexes of the heteroscorpionate ligands (3-tert-butyl-2-thio-5-methylphenyl)bis(3,5-dimethylpyrazolyl)methane (L2SH) and bis(3,5-dimethyl-pyrazolyl)(1-methyl-1-sulfanylethyl)methane (L3SH) (Figure 3).

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

(back to top)

 

Research Goals & Projects:

*    Computational experiments will commence with characterizing the hydroxide and formate complexes with the PATH ligand and the metals FeII, ZnII, CoII, and NiII.  Oxidation potentials for the complexes will be calculated to assess the redox stability of the metal.  The tendency of the metal to favor a hydroxide ligand over an aqua ligand will be addressed by computing pKb values for the hydroxide ligand. 

*    Determination of the mechanism for the 4-nitrophenyl acetate hydrolysis by the (PATH)MII-hydroxide complexes will follow.  Analysis of these results will be used to gauge the effect of the thiolate ligand in facilitating catalysis.  These calculations will also provide insight as to the cause of the inhibition in ZnII-PDF – for example, whether it results from an inability to change coordination geometry or the inability of water to replace formate as a ligand to ZnII. 

*    The next stage of the investigation will involve answering analogous questions for QM models of the enzyme active sites, which will be derived from available crystal structures.  Comparison of these results with those from the biomimetic models can be used to suggest modifications which might lead to greater reactivity in the biomimetic systems. 

*    Finally, QM/MM methods can be utilized in order to include explicitly the hydrogen-bonding environment (as well as other protein environment effects) around the coordinating cysteine and histidine residues in the calculations.  The roles of non-coordinating residues near the PDF active site can then be elucidated, with implications towards predicting the results of mutations and designing enzyme inhibitors.

 

(back to top)

 

Reading:

(1)        Chan, M. K.; Gong, W. M.; Rajagopalan, P. T. R.; Hao, B.; Tsai, C. M.; Pei, D. H. Biochemistry 1997, 36, 13904-13909.

(2)        Hao, B.; Gong, W. M.; Rajagopalan, P. T. R.; Zhou, Y.; Pei, D. H.; Chan, M. K. Biochemistry 1999, 38, 4712-4719.

(3)        Lipscomb, W. N.; Strater, N. Chem. Rev. 1996, 96, 2375-2433.

(4)        Chang, S. C.; Karambelkar, V. V.; diTargiani, R. C.; Goldberg, D. P. Inorg. Chem. 2001, 40, 194-195.

(5)        Chang, S. C.; Sommer, R. D.; Rheingold, A. L.; Goldberg, D. P. Chem. Commun. 2001, 2396-2397.

(6)        Hammes, B. S.; Carrano, C. J. Inorg. Chem. 1999, 38, 4593-4600.

 

(back to top)

 

 

(back to main research page)