Search

Alanine aminotransferase (ALT) plays significant role in biological and clinical research. In this study, a unique ALT enzyme reactor based on multifunctional polymer@magnetic nanoparticles has been constructed for the first time and the enzymolysis efficiency has been evaluated by chiral ligand exchange capillary electrophoresis technique. Poly(N-acryloxysuccinimide) has been synthesized by reversible addition-fragmentation chain transfer polymerization method and immobilized on the magnetic nanoparticles via the succinimide group in the polymer. Interestingly, the enzyme also could easily react with the succinimide group, which enables of ALT covalent bonding onto the polymer. The enzyme amount immobilized and the immobilization time have been investigated. Comparing with free ALT in solution (V max of free enzyme = 0.6 mM min -1 ), the resultant enzyme reactor has exhibited good reusability and stability, and displayed about five times enhanced enzymolysis efficiency with L-alanine as the substrate (V max of enzyme reactor = 3.4 mM min -1 ). Furthermore, the prepared enzyme reactor has been applied in ALT inhibitors screening. The enzyme reactors based on the multifunctional polymer@magnetic nanoparticles have depicted great potential in anti-liver drugs development, liver diseases study and ALT related biological process inspect., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Enzyme design is an important area of ongoing research with a broad range of applications in protein therapeutics, biocatalysis, bioengineering, and other biomedical areas; however, significant challenges exist in the design of enzymes to catalyze specific reactions of interest. Here, we develop a computational protocol using an approach that combines molecular dynamics, docking, and MM-GBSA scoring to predict the catalytic activity of enzyme variants. Our primary focuses are to understand the molecular basis of substrate recognition and binding in an S-stereoselective ω-aminotransferase (ω-AT), which naturally catalyzes the transamination of pyruvate into alanine, and to predict mutations that enhance the catalytic efficiency of the enzyme. The conversion of (R)-ethyl 5-methyl-3-oxooctanoate to (3S,5R)-ethyl 3-amino-5-methyloctanoate in the context of several ω-AT mutants was evaluated using the computational protocol developed in this work. We correctly identify the mutations that yield the greatest improvements in enzyme activity (20-60-fold improvement over wild type) and confirm that the computationally predicted structure of a highly active mutant reproduces key structural aspects of the variant, including side chain conformational changes, as determined by X-ray crystallography. Overall, the protocol developed here yields encouraging results and suggests that computational approaches can aid in the redesign of enzymes with improved catalytic efficiency.

In order to maintain proper cellular function, the metabolism of the bacterial microbiota presents several mechanisms oriented to keep a correctly balanced amino acid pool. Central components of these mechanisms are enzymes with alanine transaminase activity, pyridoxal 5'-phosphate-dependent enzymes that interconvert alanine and pyruvate, thereby allowing the precise control of alanine and glutamate concentrations, two of the most abundant amino acids in the cellular amino acid pool. Here we report the 2.11-Å crystal structure of full-length AlaA from the model organism Escherichia coli, a major bacterial alanine aminotransferase, and compare its overall structure and active site composition with detailed atomic models of two other bacterial enzymes capable of catalyzing this reaction in vivo, AlaC and valine-pyruvate aminotransferase (AvtA). Apart from a narrow entry channel to the active site, a feature of this new crystal structure is the role of an active site loop that closes in upon binding of substrate-mimicking molecules, and which has only been previously reported in a plant enzyme. Comparison of the available structures indicates that beyond superficial differences, alanine aminotransferases of diverse phylogenetic origins share a universal reaction mechanism that depends on an array of highly conserved amino acid residues and is similarly regulated by various unrelated motifs. Despite this unifying mechanism and regulation, growth competition experiments demonstrate that AlaA, AlaC and AvtA are not freely exchangeable in vivo, suggesting that their functional repertoire is not completely redundant thus providing an explanation for their independent evolutionary conservation.