Hao Kuang

Artificial metalloenzymes based on protein cavities: Exploring the effect of altering the metal ligand attachment position by site directed mutagenesis

In an effort to construct catalysts with enzyme-like properties, we are employing a small, cavity-containing protein as a scaffold for the attachment of catalytic groups. In earlier work we demonstrated that a phenanthroline ligand could be introduced into the cavity of the protein ALBP and used to catalyze ester hydrolysis. To examine the effect of positioning the phenanthroline catalyst at different locations within the protein cavity, three new constructs--Phen60, Phen72 and Phen104--were prepared. Each new conjugate was characterized by UV/vis spectroscopy, fluorescence spectroscopy, guanidine hydrochloride denaturation, gel filtration chromatography, and CD spectroscopy to confirm the preparation of the desired construct. Analysis of reactions containing Ala-OiPr showed that Phen60 catalyzed ester hydrolysis with less selectivity than ALBP-Phen while Phen72 promoted this same reaction with higher selectivity. Reactions with Tyr-OMe were catalyzed with higher selectivity by Phen60 and more rapidly by Phen104. These results demonstrate that both the rates and selectivities of hydrolysis reactions catalyzed by these constructs are dependent on the precise site of attachment of the metal ligand within the protein cavity.

The design of protein-based catalysts using semisynthetic methods

The combination of site-directed mutagenesis and chemical modification has resulted in the preparation of protein conjugates with new and useful properties. Proteins modified with metal-chelating groups are proving useful for mapping tertiary and quaternary interactions using the technique of affinity cleavage. The attachment of cofactors, including pyridoxal and pyridoxamine, has resulted in the preparation of semisynthetic transaminases that display enzyme-like properties, including enantioselectivity, substrate specificity and reaction-rate acceleration.

Effects of metal ions on the rates and enantioselectivities of reactions catalyzed by a series of semisynthetic transaminases created by site directed mutagenesis

Fatty acid binding proteins are a class of small 15 kDa proteins with a simple architecture that forms a large solvent sequestered cavity. In previous work, we demonstrated that reductive amination reactions could be performed in this cavity by covalent attachment of a pyridoxamine cofactor and that the rate, enantioselectivity and substrate specificity of these reactions could be altered by site directed mutagenesis. Herein, we show that the chemistry performed by these conjugates can be extended to include catalytic transamination and describe the effects of added metal ions on reaction rate and enantioselectivity. We conclude that metal ions can be used to increase the rate of reactions catalyzed by semisynthetic transaminases; however, the addition of metal ions can also retard the reaction rate. Furthermore, it appears that the presence of metal ions almost always results in an erosion of reaction enantioselectivity. This limits their utility as a practical means of increasing reaction rate. The results reported here, for four independent systems, should be considered in future designs of artificial transaminases.

Modulation of the rate, enantioselectivity, and substrate specificity of semisynthetic transaminases based on lipid binding proteins using site directed mutagenesis

Abstract Fatty acid binding proteins are a class of small 15 kDa proteins with a simple architecture that forms a large solvent sequestered cavity. In previous work, we demonstrated that reductive amination reactions could be performed in this cavity by covalent attachment of a pyridoxamine cofactor to the protein. Here, we report the results of experiments in which the position of pyridoxamine attachment has been varied by site directed mutagenesis. The conjugate IFABP-PX60 reacts at least 9.4-fold more rapidly than our original conjugate ALBP-PX, while IFABP-PX72 inverts the enantioselectivity of reactions (compared to ALBP-PX) and IFABP-PX104 displays very selective substrate specificty. These results indicate that site-directed mutagenesis can be used to tune the rate, enantioselectivity, and substrate specificity of semisynthetic transaminases based on fatty acid binding proteins.

Synthesis of a cationic pyridoxamine conjugation reagent and application to the mechanistic analysis of an artificial transaminase

An N-methylated, cationic pyridoxamine conjugation reagent was synthesized and tethered via a disulfide bond to a cysteine residue inside the cavity of intestinal fatty acid binding protein. The conjugate was characterized and the kinetic parameters compared to its nonmethylated pyridoxamine analogue. Kinetic isotope effects were used for further mechanistic analysis. Taken together, these experiments suggest that a step distinct from deprotonation of the ketimine in the pyridoxamine to pyridoxal reaction is what limits the rate of the artificial transaminase IFABP-Px. However, the internal energetics of reactions catalyzed by the conjugate containing the N-methylated cofactor appear to be different suggesting that the MPx reagent will be useful in future experiments designed to alter the catalytic properties of semisynthetic transaminases.

Converting a fatty acid binding protein to an artificial transaminase : novel catalysts by chemical and genetic modification of a protein cavity

Abstract Despite the widespread use of enzymes in synthetic applications, their “native” characteristics are often insufficient for many chemical transformations. To meet this challenge we have used protein cavities for the design of new biocatalysts. A pyridoxamine derivative (PX) was chemically tethered within the spacious cavity of intestinal fatty acid binding protein (IFABP). The cysteine residue, which anchors the cofactor of the artificial transaminase IFABP-PX, can be placed in different regions by site-directed mutagenesis. Catalytic reactions with high enantioselectivities (up to 94% ee) and varying substrate specificity of the transamination of α-keto and amino acids were achieved. IFABP-PX mutants were further optimized by introducing lysine residues in order to mimic the active site of native transaminases.

Semisynthetic approaches for the design of proteins with catalytic activity using fatty acid binding protein as a scaffold

The objective of the work described here is to develop enanatioselective catalysts that are based on protein cavities. This approach for catalyst design combines elements of hostguest chemistry with a highly flexible protein scaffold that can be manipulated by both chemical modification and recombinant DNA methods [1-4]. The ability to prepare such catalysts could have a significant impact on the manufacture of a wide variety of specialty chemicals. In our earlier work, we demonstrated that a protein cavity (Intestinal Fatty Acid Binding Protein, IFABP) could be chemically modified with catalytic groups to prepare constructs that performed enantioselective reactions [5,6]. We also showed that the enantioselectivity, rate and substrate specificity could be altered by varying the point of attachment between the catalytic group and the protein [7,8]. The structures of two of these constructs were solved by X-ray diffraction methods; the structure of ALBP-PX (a related FABP) is shown below in Fig. 1 [9]. Here, we describe three recent efforts directed towards improving the efficiency of FABP-based conjugates. These include the use of a modified cofactor (MPX), the preparation of a modified protein scaffold (helixless) and the incorporation of active site lysine residues via site directed mutagenesis.