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Hydrolytic catalysis and structural stabilization in a designed metalloprotein.

ABSTRACT: Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions-a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displays catalytic activity that compares well with several characteristic reactions of natural enzymes. It catalyses p-nitrophenyl acetate (pNPA) hydrolysis with an efficiency only ~100-fold less than that of human carbonic anhydrase (CA)II and at least 550-fold better than comparable synthetic complexes. Similarly, CO(2) hydration occurs with an efficiency within ~500-fold of CAII. Although histidine residues in the absence of Zn(II) exhibit pNPA hydrolysis, miniscule apopeptide activity is observed for CO(2) hydration. The kinetic and structural analysis of this first de novo designed hydrolytic metalloenzyme reveals necessary design features for future metalloenzymes containing one or more metals.

SUBMITTER: Zastrow ML

PROVIDER: S-EPMC3270697 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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Hydrolytic catalysis and structural stabilization in a designed metalloprotein.

Zastrow Melissa L ML Peacock Anna F A AF Stuckey Jeanne A JA Pecoraro Vincent L VL

Nature chemistry 20111127 2

Metal ions are an important part of many natural proteins, providing structural, catalytic and electron transfer functions. Reproducing these functions in a designed protein is the ultimate challenge to our understanding of them. Here, we present an artificial metallohydrolase, which has been shown by X-ray crystallography to contain two different metal ions-a Zn(II) ion, which is important for catalytic activity, and a Hg(II) ion, which provides structural stability. This metallohydrolase displ ...[more]

PMID: 22270627

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Project description:The relationship between structure and function has long been one of the major points of investigation in Biophysics. Understanding how much, or how little, of a protein's often complicated structure is necessary for its function can lead to directed therapeutic strategies and would allow one to design proteins for specific desired functions. Studying protein function by de novo design builds the functionality from the ground up in a completely unrelated and noncoded protein scaffold. Our lab has used this strategy to study heavy and transition metal binding within the TRI family of three stranded coiled coil (3SCC) constructs to understand coordination geometry and metalloenzyme catalytic control within a protein environment. These peptides contain hydrophobic layers within the interior of the 3SCC, which one can mutate to metal binding residues to create a minimal metal binding site, while solid phase synthesis allows our lab to easily incorporate a number of noncoded amino acids including d enantiomers of binding or secondary coordination sphere amino acids, penicillamine, or methylated versions of histidine. Our studies of Cd(II) binding to Cys3 environments have determined, largely through the use of 113Cd NMR and 111mCd PAC, that the coordination environment around a heavy metal can be controlled by incorporating noncoded amino acids in either the primary or secondary coordination spheres. We found mutating the metal binding amino acids to l-Pen can enforce trigonal Cd(II)S3 geometry exclusively compared to the mixed coordination determined for l-Cys coordination. The same result can be achieved with secondary sphere mutations as well by incorporating d-Leu above a Cys3. We hypothesize this latter effect is due to the increased steric packing above the metal binding site that occurs when the l-Leu oriented toward the N-terminus of the scaffold is mutated to d-Leu and oriented toward the C-terminus. Mutating the layer below Cys3 to d-Leu instead formed a mixed 4- and 5-coordinate Cd(II)S3(H2O) and Cd(II)S3(H2O)2 construct as steric bulk was decreased below the metal binding site. We have also applied noncoded amino acids to metalloenzyme systems by incorporating His residues that are methylated at the δ- or ε-nitrogen to enforce Cu(I) ligation to the opposite open nitrogen of His and found a 2 orders of magnitude increased catalytic efficiency for nitrite reductase activity with ε-nitrogen coordination compared to δ-nitrogen. These results exemplify the ability to tune coordination environment and catalytic efficiency within a de novo scaffold as well as the utility of noncoded amino acids to increase the chemist's toolbox. By furthering our understanding of metalloprotein design one could envision, through our use of amino acids not normally available to nature, that protein design laboratories will soon be capable of outperforming the native systems previously used as their benchmark of successful design. The ability to design proteins at this level would have far reaching and exciting benefits within various fields including medical and industrial applications.

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Hydrolytic catalysis and structural stabilization in a designed metalloprotein.

Publications

Hydrolytic catalysis and structural stabilization in a designed metalloprotein.

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