Project description:Kynureninase is a member of a large family of catalytically diverse but structurally homologous pyridoxal 5'-phosphate (PLP) dependent enzymes known as the aspartate aminotransferase superfamily or alpha-family. The Homo sapiens and other eukaryotic constitutive kynureninases preferentially catalyze the hydrolytic cleavage of 3-hydroxy-l-kynurenine to produce 3-hydroxyanthranilate and l-alanine, while l-kynurenine is the substrate of many prokaryotic inducible kynureninases. The human enzyme was cloned with an N-terminal hexahistidine tag, expressed, and purified from a bacterial expression system using Ni metal ion affinity chromatography. Kinetic characterization of the recombinant enzyme reveals classic Michaelis-Menten behavior, with a Km of 28.3 +/- 1.9 microM and a specific activity of 1.75 micromol min-1 mg-1 for 3-hydroxy-dl-kynurenine. Crystals of recombinant kynureninase that diffracted to 2.0 A were obtained, and the atomic structure of the PLP-bound holoenzyme was determined by molecular replacement using the Pseudomonas fluorescens kynureninase structure (PDB entry 1qz9) as the phasing model. A structural superposition with the P. fluorescens kynureninase revealed that these two structures resemble the "open" and "closed" conformations of aspartate aminotransferase. The comparison illustrates the dynamic nature of these proteins' small domains and reveals a role for Arg-434 similar to its role in other AAT alpha-family members. Docking of 3-hydroxy-l-kynurenine into the human kynureninase active site suggests that Asn-333 and His-102 are involved in substrate binding and molecular discrimination between inducible and constitutive kynureninase substrates.
Project description:Post-transcriptional regulation is crucial to shape gene expression. During the Maternal-to-Zygotic transition (MZT), thousands of maternal transcripts are regulated upon fertili-zation and genome activation. Transcript stability can be influenced by cis-elements and trans-factors, but how these inputs are integrated to determine the overall mRNA stability is unclear. Here, we show that most transcripts are under combinatorial regulation by multiple decay pathways. Characterization of the cis-regulatory motifs revealed that nu-cleotide composition bias characteristic of 3’-UTRs poly-U is associated with mRNA stability. In contrast, miR-430, CCUC, CUGC, elements appeared as the main destabiliz-ing motifs, with miR-430 and UAUUUAU (ARE) sequences causing mRNA deadenyla-tion depending on the activation of the genome. We comprehensively identify RNA-protein interactions across the transcriptome during MZT, and their associated regulatory activity. We find that poly-U binding proteins are preferentially associated with 3’-UTR sequences and stabilizing motifs. Analysis of differentially regulated regions revealed antagonistic sequence contexts for poly-C and poly-U binding proteins that shape protein binding and magnitude of regulation across the transcriptome. Finally, we integrate these regulatory motifs into a machine learning model, able to predict the stability of mRNA reporters in vivo. Our findings reveal how mechanisms of post-transcriptional regulation are coordinated to direct changes in mRNA stability within the early embryo.