The structure of the periplasmic nickel-binding protein NikA provides insights for artificial metalloenzyme design

Abstract

Understanding the interaction of a protein with a relevant ligand is crucial for the design of an artificial metalloenzyme. Our own interest is focused on the synthesis of artificial monooxygenases. In an initial effort, we have used the periplasmic nickel-binding protein NikA from Escherichia coli and iron complexes in which N(2)Py(2) ligands (where Py is pyridine) have been varied in terms of charge, aromaticity, and size. Six "NikA/iron complex" hybrids have been characterized by X-ray crystallography, and their interactions and solution properties have been studied. The hybrids are stable as indicated by their K (d) values, which are all in the micromolar range. The X-ray structures show that the ligands interact with NikA through salt bridges with arginine residues and π-stacking with a tryptophan residue. We have further characterized these interactions using quantum mechanical calculations and determined that weak CH/π hydrogen bonds finely modulate the stability differences between hybrids. We emphasize the important role of the tryptophan residues. Thus, our study aims at the complete characterization of the factors that condition the interaction of an artificial ligand and a protein and their implications for catalysis. Besides its potential usefulness in the synthesis of artificial monooxygenases, our approach should be generally applicable in the field of artificial metalloenzymes.

Structural Basis for Enantioselectivity in the Transfer Hydrogenation of a Ketone Catalyzed by an Artificial Metalloenzyme

“…Orientations that fitted the electron density of the carbonyl moiety and one of the 2,2‐dipyridylamine rings (Figure 3a) were retained and could be divided into three families (Figure 3b–d). Weak CH‐π type interactions (1.5–2.5 kcal/mol) between the π orbitals of the aromatic rings of complex 1 and the protein side chains23 may have contributed to the stabilization of the complex 1 head in these orientations. The reaction catalyzed by the Rh III d 6 piano‐stool complexes used in this study involves the outer‐sphere attack of the metal‐bound hydride on the substrate's ketone group.…”

Protein Design: Toward Functional Metalloenzymes

“…Furthermore, a series of iron complexes with either N , N ′-dimethyl- N , N ′-bis(2-pyridylmethyl)ethane-1,2-diamine (BPMEN) ligand or N , N ′-bis(2-pyridylmethyl)- N , N ′-dimethyl- trans -1,2-diaminocyclohexane (BPMCN) ligand derivatives were incorporated into apo-NikA. 581 These complexes had K d values in the micromolar range, with complexes A and B shown in Scheme 14 having the highest and the lowest affinities, respectively. They obtained crystal structures of some of hybrid proteins, which revealed H-bonding interactions, as well as CH/ π H-bonds that are essential in forming these NikA complexes.…”

Heavy Metal Removal by Bioaccumulation Using Genetically Engineered Microorganisms

“…From this, there may be ancient HM transporters with promiscuous transport activity that could serve as templates in directed evolution with HM riboswitches to evolve new HM transporters. Storage systems may also benefit from protein engineering efforts where researchers have intensively focused on engineering metal-binding sites (Cherrier et al, 2012 ; Valasatava et al, 2015 ; Akcapinar and Sezerman, 2017 ). Such efforts may use existing MBPs as templates to produce “cysteine-free” storage proteins that are intrinsically soluble.…”