No abstract

Significance Protein flexibility has been recognized as a key contributor to enzyme evolution and catalytic activity. Several studies have illustrated how amino acid substitutions that affect protein flexibility can impact catalysis. However, it is unknown whether structural information regarding conformational flexibility can be exploited for directed evolution of enzymes with higher catalytic activity. Using as a model human kynureninase, an enzyme with important therapeutic implications in cancer immunotherapy, we show that mutagenesis of residues exclusively located within high B-factor regions distal to the active site resulted in a variant with markedly enhanced catalytic activity for its nonpreferred substrate, kynurenine. Our results suggest that modulation of intrinsic flexibility through mutagenesis of remote flexible regions constitutes a promising strategy for directed enzyme evolution.

Kynureninases (KYNases) are enzymes that play a key role in tryptophan catabolism through the degradation of intermediate kynurenine and 3′-hydroxy-kynurenine metabolites (KYN and OH-KYN, respectively). Bacterial KYNases exhibit high catalytic efficiency toward KYN and moderate activity toward OH-KYN, whereas animal KYNases are highly selective for OH-KYN, exhibiting only minimal activity toward the smaller KYN substrate. These differences reflect divergent pathways for KYN and OH-KYN utilization in the respective kingdoms. We examined the Homo sapiens and Pseudomonas fluorescens KYNases (HsKYNase and PfKYNase respectively) using pre-steady-state and hydrogen–deuterium exchange mass spectrometry (HDX-MS) methodologies. We discovered that the activity of HsKYNase critically depends on formation of hydrogen bonds with the hydroxyl group of OH-KYN to stabilize the entire active site and allow productive substrate turnover. With the preferred OH-KYN substrate, stabilization is observed at the substrate-binding site and the region surrounding the PLP cofactor. With the nonpreferred KYN substrate, less stabilization occurs, revealing a direct correlation with activity. This correlation holds true for PfKYNases; however there is only a modest stabilization at the substrate-binding site, suggesting that substrate discrimination is simply achieved by steric hindrance. We speculate that eukaryotic KYNases use dynamic mobility as a mechanism of substrate specificity to commit OH-KYN to nicotinamide synthesis and avoid futile hydrolysis of KYN. These findings have important ramifications for the engineering of HsKynase with high KYN activity as required for clinical applications in cancer immunotherapy. Our study shows how homologous enzymes with conserved active sites can use dynamics to discriminate between two highly similar substrates.

No abstract

Activins are members of the Transforming Growth Factor‐β superfamily. This superfamily of secreted growth factors activates transmembrane serine‐threonine kinase receptors, thereby stimulating Smad‐mediated intracellular signaling cascades. These cascades alter the gene expression profile of the target cells. Follistatin (FST) is a protein antagonist that directly binds to activins and other TGF‐β‐like proteins, preventing their interaction with cell‐surface receptors. Alternative splicing and proteolysis result in three isoforms of FST which differ in their C‐terminal regions. The three FST isoforms have different affinities for activins, as well as the extracellular matrix component heparan sulfate. We are focused on two FST isoforms, FST315 and FST288. FST315 contains an acidic C‐terminal region that is absent in FST288. Crystal structures of FST315 and FST288 with activin A are unable to explain the basis for isoform‐specific effects. The structures are similar and the acidic C‐terminal region of FST315 is not observed in the electron density. To identify differences between FST315 and FST288, we have used hydrogen/deuterium exchange coupled to mass spectrometry. We compare exchange of FST315 and FST288, both alone and in complex with activin A. The results will improve our molecular understanding of how FST isoforms regulate signaling by activin A.Support or Funding InformationStartup funds to S. D'Arcy from The University of Texas at Dallas.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.