Understanding enzymes specificities as a tool for cofactor engineering

S-adenosylmethionine (SAM) is the second most abundant cofactor in nature after adenosyl triphosphate (ATP). SAM is synthesized in a cell by Methionine adenosyltransferase (MAT) using ATP and methionine and it is used as a methyl donor to methylate different substrates (DNA, protein, RNA, small molecules) by methyltransferases (Mtases). The methylation is a key process for cellular regulation and aberrant methylation associated with the disease condition. We are aiming at engineering two-steps pathway for an orthogonal cofactor, for this purpose we first decided to explore the promiscuities of the nucleotide base of ATP for two enzymes (MAT and Mtases). To find good candidates for these engineering we expressed and purified MAT from different organisms. We found MAT from specific organisms are promiscuous for the new nucleotide-based cofactor. It is very interesting that certain MAT is promiscuous for nucleotides-based cofactor but are these newly formed cofactors also promiscuous for the methyltransferase (Mtases) who is the main user of theses cofactors? Further, we have also investigated promiscuity of the DNA methyltransferase (Mtases) from bacteria. Overall these findings lay the foundation for our engineering studies and hint at the evolution of these enzymes. References: Gade, M., Villar-Briones, A.; Laurino, P. “Understanding Enzymes Promiscuity for Novel Cofactors” Submitted. G. L. Cantoni, J. Am. Chem. Soc. 1952, 74, 2942-2943. O. Khersonsky, D. S. Tawfik, Annu Rev Biochem 2010, 79, 471-505

Rossmann-Fold Methyltransferases: Taking a “β-Turn” around Their Cofactor, S-Adenosylmethionine

Methyltransferases (MTases) are superfamilies of enzymes that catalyze the transfer of a methyl group from S-adenosylmethionine (SAM), a nucleoside-based cofactor, to a wide variety of substrates such as DNA, RNA, proteins, small molecules, and lipids. Depending upon their structural features, the MTases can be further classified into different classes; we consider exclusively the largest class of MTases, the Rossmann-fold MTases. It has been shown that the nucleoside cofactor-binding Rossmann enzymes, particularly the nicotinamide adenine dinucleotide (NAD)-, flavin adenine dinucleotide (FAD)-, and SAM-binding MTases enzymes, share common binding motifs that include a Gly-rich loop region that interacts with the cofactor and a highly conserved acidic residue (Asp/Glu) that interacts with the ribose moiety of the cofactor. Here, we observe that the Gly-rich loop region of the Rossmann MTases adapts a specific type II′ β-turn in the proximity of the cofactor (<4 Å), and it appears to be a key feature of these superfamilies. Additionally, we demonstrate that the conservation of this β-turn could play a critical role in the enzyme–cofactor interaction, thereby shedding new light on the structural conformation of the Gly-rich loop region from Rossmann MTases

Implications of divergence of methionine adenosyltransferase in archaea

Methionine adenosyltransferase (MAT) catalyzes the biosynthesis of S-adenosylmethionine from L-methionine and adenosine triphosphate. MAT enzymes are ancient, believed to share a common ancestor, and are highly conserved in all three domains of life. However, the sequences of archaeal MATs show considerable divergence compared to their bacterial and eukaryotic counterparts. Furthermore, the structural and functional significance of this sequence divergence are not well understood. In the present study, we employed structural analysis and ancestral sequence reconstruction (ASR) to investigate archaeal MAT divergence. We observed that the dimer interface containing the active site (which is usually well-conserved) diverged considerably between the bacterial/eukaryotic MATs and archaeal MAT. A detailed investigation of the available structures supports the sequence analysis outcome: the protein domains and subdomains of bacterial and eukaryotic MAT are more similar than those of archaea. Finally, we resurrected archaeal MAT ancestors. Interestingly, archaeal MAT ancestors show substrate specificity, which is lost during evolution. This observation supports the hypothesis of a common MAT ancestor for the three domains of life. In conclusion, we have demonstrated that archaeal MAT is an ideal system for studying an enzyme family that evolved differently in one domain compared to others while maintaining the same catalytic activity

<span style="font-size:15.0pt;mso-bidi-font-weight:bold" lang="EN-US">Cu^II-exchanged montmorillonite K10 clay-catalyzed direct carboxylation of terminal alkynes with carbon dioxide </span>

1325-1329A new, simple and straight<b style="mso-bidi-font-weight: normal">-forward protocol for direct carboxylation of terminal alkynes <span style="font-size:12.0pt;mso-bidi-font-size: 9.0pt" lang="EN-GB">has been developed<span style="mso-bidi-font-size: 9.0pt" lang="EN-GB"> using <span style="font-size:12.0pt; mso-bidi-font-size:9.0pt" lang="EN-GB">CuII-montmorillonite K10 clay as a heterogeneous catalyst and CO2 as the C1 carbon feedstock. Also coupling of terminal alkynes with CO2 (1 atm) in the presence of alkyl halides has been achieved under the same reaction conditions, thereby providing access to a variety of functionalized alkyl-2-alkynoates in high yields. </span

Modeling Glyco-Collagen Conjugates Using a Host–Guest Strategy To Alter Phenotypic Cell Migration and <i>in Vivo</i> Wound Healing

The constructs and study of combinatorial libraries of structurally defined homologous extracellular matrix (ECM) glycopeptides can significantly accelerate the identification of cell surface markers involved in a variety of physiological and pathological processes. Herein, we present a simple and reliable host–guest approach to design a high-throughput glyco-collagen library to modulate the primary and secondary cell line migration process. 4-Amidoadamantyl-substituted collagen peptides and β-cyclodextrin appended with mono- or disaccharides were used to construct self-assembled glyco-collagen conjugates (GCCs), which were found to be thermally stable, with triple-helix structures and nanoneedles-like morphologies that altered cell migration processes. We also investigated the glycopeptide’s mechanisms of action, which included interactions with integrins and cell signaling kinases. Finally, we report murine wound models to demonstrate the real-time application of GCCs. As a result of our observations, we claim that the host–guest model of ECM glycopeptides offers an effective tool to expedite identification of specific glycopeptides to manipulate cell morphogenesis, cell differentiation metastatic processes, and their biomedical applications