Designing Hydrolytic Zinc Metalloenzymes

Abstract

Zinc is an essential element required for the function of more than 300 enzymes spanning all classes. Despite years of dedicated study, questions regarding the connections between primary and secondary metal ligands and protein structure and function remain unanswered, despite numerous mechanistic, structural, biochemical, and synthetic model studies. Protein design is a powerful strategy for reproducing native metal sites that may be applied to answering some of these questions and subsequently generating novel zinc enzymes. From examination of the earliest design studies introducing simple Zn(II)-binding sites into de novo and natural protein scaffolds to current studies involving the preparation of efficient hydrolytic zinc sites, it is increasingly likely that protein design will achieve reaction rates previously thought possible only for native enzymes. This Current Topic will review the design and redesign of Zn(II)-binding sites in de novo-designed proteins and native protein scaffolds toward the preparation of catalytic hydrolytic sites. After discussing the preparation of Zn(II)-binding sites in various scaffolds, we will describe relevant examples for reengineering existing zinc sites to generate new or altered catalytic activities. Then, we will describe our work on the preparation of a de novo-designed hydrolytic zinc site in detail and present comparisons to related designed zinc sites. Collectively, these studies demonstrate the significant progress being made toward building zinc metalloenzymes from the bottom up.

Protein Design: Toward Functional Metalloenzymes

“…212,213 This de novo designed 3SCC contains two metal sites, one for structural stabilization (HgS 3 ) and the second for hydrolytic activity (ZnN 3 O) within just a few hundred-fold of the native CA. Although the design of this metalloprotein has been previously reviewed, 23,822 it is appropriate to summarize the initial work before reviewing more recent developments.…”

Coordination complexes and biomolecules: A wise wedding for catalysis upgrade

“…These systems, first developed to study the binding of toxic heavy metals [43], have required huge effort to control the geometry of the binding site [44]. These systems were then transposed for Cu [45,46] and Zn coordination [41,47] showing promising results as artificial metalloenzymes [48][49][50] such as nitrite reductase [46] and hydrolase [47] or hydratase [41].…”

Computational strategies for the design of new enzymatic functions

“…143,144,145 X-ray crystallography showed that the designed protein contained two different metal ions; Zn(II), which is important for catalysis, and a Hg(II) ion, which provides structural stability. In these hydrolytic zinc metalloenzymes, it was demonstrated the influence of location of the active site formed by the metal and three histidine residues in a three-stranded coiled-coil oligopeptide.…”