DARPin-Based Crystallization Chaperones Exploit Molecular Geometry as a Screening Dimension in Protein Crystallography.

DARPin libraries, based on a Designed Ankyrin Repeat Protein consensus framework, are a rich source of binding partners for a wide variety of proteins. Their modular structure, stability, ease of in vitro selection and high production yields make DARPins an ideal starting point for further engineering. The X-ray structures of around 30 different DARPin complexes demonstrate their ability to facilitate crystallization of their target proteins by restricting flexibility and preventing undesired interactions of the target molecule. However, their small size (18 kDa), very hydrophilic surface and repetitive structure can limit the DARPins' ability to provide essential crystal contacts and their usefulness as a search model for addressing the crystallographic phase problem in molecular replacement. To optimize DARPins for their application as crystallization chaperones, rigid domain-domain fusions of the DARPins to larger proteins, proven to yield high-resolution crystal structures, were generated. These fusions were designed in such a way that they affect only one of the terminal capping repeats of the DARPin and do not interfere with residues involved in target binding, allowing to exchange at will the binding specificities of the DARPin in the fusion construct. As a proof of principle, we designed rigid fusions of a stabilized version of Escherichia coli TEM-1 ß-lactamase to the C-terminal capping repeat of various DARPins in six different relative domain orientations. Five crystal structures representing four different fusion constructs, alone or in complex with the cognate target, show the predicted relative domain orientations and prove the validity of the concept.

Metabolism of ß-valine via a CoA-dependent ammonia lyase pathway.

Pseudomonas species strain SBV1 can rapidly grow on medium containing ß-valine as a sole nitrogen source. The tertiary amine feature of ß-valine prevents direct deamination reactions catalyzed by aminotransferases, amino acid dehydrogenases, and amino acid oxidases. However, lyase- or aminomutase-mediated conversions would be possible. To identify enzymes involved in the degradation of ß-valine, a PsSBV1 gene library was prepared and used to complement the ß-valine growth deficiency of a closely related Pseudomonas strain. This resulted in the identification of a gene encoding ß-valinyl-coenzyme A ligase (BvaA) and two genes encoding ß-valinyl-CoA ammonia lyases (BvaB1 and BvaB2). The BvaA protein demonstrated high sequence identity to several known phenylacetate CoA ligases. Purified BvaA enzyme did not convert phenyl acetic acid but was able to activate ß-valine in an adenosine triphosphate (ATP)- and CoA-dependent manner. The substrate range of the enzyme appears to be narrow, converting only ß-valine and to a lesser extent, 3-aminobutyrate and ß-alanine. Characterization of BvaB1 and BvaB2 revealed that both enzymes were able to deaminate ß-valinyl-CoA to produce 3-methylcrotonyl-CoA, a common intermediate in the leucine degradation pathway. Interestingly, BvaB1 and BvaB2 demonstrated no significant sequence identity to known CoA-dependent ammonia lyases, suggesting they belong to a new family of enzymes. BLAST searches revealed that BvaB1 and BvaB2 show high sequence identity to each other and to several enoyl-CoA hydratases, a class of enzymes that catalyze a similar reaction with water instead of amine as the leaving group.

Ironing out their differences: dissecting the structural determinants of a phenylalanine aminomutase and ammonia lyase.

Deciphering the structural features that functionally separate ammonia lyases from aminomutases is of interest because it may allow for the engineering of more efficient aminomutases for the synthesis of unnatural amino acids (e.g., ß-amino acids). However, this has proved to be a major challenge that involves understanding the factors that influence their activity and regioselectivity differences. Herein, we report evidence of a structural determinant that dictates the activity differences between a phenylalanine ammonia lyase (PAL) and aminomutase (PAM). An inner loop region that closes the active sites of both PAM and PAL was mutated within PAM (PAM residues 77-97) in a stepwise approach to study the effects when the equivalent residue(s) found in the PAL loop were introduced into the PAM loop. Almost all of the single loop mutations triggered a lyase phenotype in PAM. Experimental and computational evidence suggest that the induced lyase features result from inner loop mobility enhancements, which are possibly caused by a 310-helix cluster, flanking α-helices, and hydrophobic interactions. These findings pinpoint the inner loop as a structural determinant of the lyase and mutase activities of PAM.

Priming ammonia lyases and aminomutases for industrial and therapeutic applications.

Ammonia lyases (AL) and aminomutases (AM) are emerging in green synthetic routes to chiral amines and an AL is being explored as an enzyme therapeutic for treating phenylketonuria and cancer. Although the restricted substrate range of the wild-type enzymes limits their widespread application, the non-reliance on external cofactors and direct functionalization of an olefinic bond make ammonia lyases attractive biocatalysts for use in the synthesis of natural and non-natural amino acids, including ß-amino acids. The approach of combining structure-guided enzyme engineering with efficient mutant library screening has extended the synthetic scope of these enzymes in recent years and has resolved important mechanistic issues for AMs and ALs, including those containing the MIO (4-methylideneimidazole-5-one) internal cofactor.

Mechanism-inspired engineering of phenylalanine aminomutase for enhanced ß-regioselective asymmetric amination of cinnamates.

Turn to switch: A mutant of phenylalanine aminomutase was engineered that can catalyze the regioselective amination of cinnamate derivatives (see scheme, red) to, for example, ß-amino acids. This regioselectivity, along with the X-ray crystal structures, suggests two distinct carboxylate binding modes differentiated by C(ß)-C(ipso) bond rotation, which determines if ß- (see scheme) or α-addition takes place.

Aminomutases: mechanistic diversity, biotechnological applications and future perspectives.

Aminomutases carry out the chemically challenging exchange of a hydrogen atom and an amine substituent present on neighboring carbon atoms. In recent years, aminomutases have been intensively investigated for their biophysical, structural and mechanistic characteristics. The reactions catalyzed by these enzymes have considerable potential for biotechnological applications. Here, we present an overview of this diverse group of enzymes, with a focus on enzymatic mechanisms and recent developments in their use in applied biocatalysis.