New Tricks for Old Proteins: Single Mutations in a Nonenzymatic Protein Give Rise to Various Enzymatic Activities.

Design of a new catalytic function in proteins, apart from its inherent practical value, is important for fundamental understanding of enzymatic activity. Using a computationally inexpensive, minimalistic approach that focuses on introducing a single highly reactive residue into proteins to achieve catalysis we converted a 74-residue-long C-terminal domain of calmodulin into an efficient esterase. The catalytic efficiency of the resulting stereoselective, allosterically regulated catalyst, nicknamed AlleyCatE, is higher than that of any previously reported de novo designed esterases. The simplicity of our design protocol should complement and expand the capabilities of current state-of-art approaches to protein design. These results show that even a small nonenzymatic protein can efficiently attain catalytic activities in various reactions (Kemp elimination, ester hydrolysis, retroaldol reaction) as a result of a single mutation. In other words, proteins can be just one mutation away from becoming entry points for subsequent evolution.

Design of an allosterically regulated retroaldolase.

We employed a minimalist approach for design of an allosterically controlled retroaldolase. Introduction of a single lysine residue into the nonenzymatic protein calmodulin led to a 15,000-fold increase in the second order rate constant for retroaldol reaction with methodol as a substrate. The resulting catalyst AlleyCatR is active enough for subsequent directed evolution in crude cell bacterial lysates. AlleyCatR's activity is allosterically regulated by Ca(2+) ions. No catalysis is observed in the absence of the metal ion. The increase in catalytic activity originates from the hydrophobic interaction of the substrate (∼2000-fold) and the change in the apparent pKa of the active lysine residue.

Crystal structure of {µ-6,6'-dimeth-oxy-2,2'-[ethane-1,2-diylbis(nitrilo-methanylyl-idene)]diphenolato}(meth-anol)(nitrato)nickel(II)sodium.

In the molecular structure of the title compound, [NaNi(C18H18N2O4)(NO3)(CH3OH)], the Ni(2+) ion has a slightly distorted square-planar coordination environment defined by two N and two O atoms which belong to a Schiff base ligand, viz. 6,6'-dimeth-oxy-2,2'-[ethane-1,2-diylbis(nitrilo-methanylyl-idene)]diphenolate. Seven O atoms form the coordination environment of the Na(+) ion: four from the Schiff base ligand, two from a bidentate chelating nitrate anion and one O atom from a coordinating methanol mol-ecule. In the crystal, the bimetallic complexes are assembled into chains along the b-axis direction via weak C-H⋯O hydrogen-bond inter-actions. Neighbouring chains are in turn connected through bifurcated O-H⋯O hydrogen bonds that involve the coordinating methanol mol-ecules and the nitrate anions, and through π-π stacking inter-actions between phenyl rings of neighbouring mol-ecules.

{Dimeth-yl [(phenyl-sulfon-yl)amido-]phosphato-κ(2) O,O'}bis-(tri-phenylphosphane-κP)copper(I).

In the title complex, [Cu(C8H11NO5PS)(C18H15P)2], the Cu(I) ion is coordinated by two tri-phenyl-phosphane mol-ecules and two O atoms of the chelating dimeth-yl(phenyl-sulfon-yl)amido-phosphate anion, generating a squashed CuO2P2 tetrahedron. In the six-membered chelate ring, the Cu, P and O atoms are almost coplanar (r.m.s. deviation = 0.024 Å), with the N and S atoms displaced in the same direction, by 0.708 (5) and 0.429 (2) Å, respectively.

Short peptides self-assemble to produce catalytic amyloids.

Enzymes fold into unique three-dimensional structures, which underlie their remarkable catalytic properties. The requirement to adopt a stable, folded conformation is likely to contribute to their relatively large size (>10,000 Da). However, much shorter peptides can achieve well-defined conformations through the formation of amyloid fibrils. To test whether short amyloid-forming peptides might in fact be capable of enzyme-like catalysis, we designed a series of seven-residue peptides that act as Zn(2+)-dependent esterases. Zn(2+) helps stabilize the fibril formation, while also acting as a cofactor to catalyse acyl ester hydrolysis. These results indicate that prion-like fibrils are able to not only catalyse their own formation, but they can also catalyse chemical reactions. Thus, they might have served as intermediates in the evolution of modern-day enzymes. These results also have implications for the design of self-assembling nanostructured catalysts including ones containing a variety of biological and non-biological metal ions.

Reprogramming EF-hands for design of catalytically amplified lanthanide sensors.

We recently reported that a computationally designed catalyst nicknamed AlleyCat facilitates C-H proton abstraction in Kemp elimination at neutral pH in a selective and calcium-dependent fashion by a factor of approximately 100,000 (Korendovych et al. in Proc. Natl. Acad. Sci. USA 108:6823, 2011). Kemp elimination produced a colored product that can be easily read out, thus making AlleyCat a catalytically amplified metal sensor for calcium. Here we report that metal-binding EF-hand motifs in AlleyCat could be redesigned to incorporate trivalent metal ions without significant loss of catalytic activity. Mutation of a single neutral residue at position 9 of each of the EF-hands to glutamate results in almost a two orders of magnitude improvement of selectivity for trivalent metal ions over calcium. Development of this new lanthanide-dependent switchable Kemp eliminase, named CuSeCat EE, provides the foundation for further selectivity improvement and broadening the scope of the repertoire of metals for sensing. A concerted effort in the design of switchable enzymes has many environmental, sensing, and metal ion tracking applications.

N-[Bis(dimethyl-amino)phosphinoyl]-2,2,2-trichloro-acetamide.

In the title compound, C(6)H(13)Cl(3)N(3)O(2)P or CCl(3)C(O)NHP(O)(N(CH(3))(2)), the phosphinoyl group is synclinal to the carbonyl group and acts as an acceptor for an inter-molecular N-H⋯O hydrogen bond from the amide group as the donor.

Tris{N-[bis-(dimethyl-amino)phosphino-yl]-2,2,2-trichloro-acetamido}(triphenyl-phosphine oxide)holmium(III).

In the title compound, [Ho(C(6)H(12)Cl(3)N(3)O(2)P)(3)(C(18)H(15)OP)], the Ho(III) ion is surrounded by six O atoms from the three bidentate N-[bis-(dimethyl-amino)phosphino-yl]-2,2,2-trichloro-acetamido ligands (L(-)) and by one O atom from the triphenyl-phosphine oxide ligand, with the formation of a distorted monocapped octa-hedron. In one ligand L(-), the trichloro-methyl group is rotationally disordered between two orientations in a 1:1 ratio, while two dimethyl-amino groups in another ligand L(-) are disordered between two conformations, each with the same 1:1 ratio.

Bis{N-[bis-(pyrrolidin-1-yl)phosphor-yl]-2,2,2-trichloro-acetamide}di-nitrato-dioxidouranium(VI).

The crystal structure of the title compound, [U(NO(3))(2)O(2)(C(10)H(17)Cl(3)N(3)O(2)P)(2)], is composed of centrosymmetric [UO(2)(L)(2)(NO(3))(2)] mol-ecules {L is N-[bis-(pyrrolidin-1-yl)phosphor-yl]-2,2,2-trichloro-acetamide, C(10)H(17)Cl(3)N(3)O(2)P}. The U(VI) ion, located on an inversion center, is eight-coordinated with axial oxido ligands and six equatorial oxygen atoms of the phosphoryl and nitrate groups in a slightly distorted hexa-gonal-bipyramidal geometry. One of the pyrrolidine fragments in the ligand is disordered over two conformation (occupancy ratio 0.58:0.42). Intra-molecular N-H⋯O hydrogen bonds between the amine and nitrate groups are found.