RESUMEN

This study delves into the functional intricacies of lipoate ligase A (LplA), an enzyme showing great promise in bioconjugation due to its unique capacity for introducing azido groups into proteins without requiring a genetic tag. We aimed to enhance the understanding of LplA's functionality, particularly its substrate tolerance and the reliability of various analytical techniques. A pivotal aspect of our approach was incorporating azido groups into a range of proteins, followed by the addition of the fluorescent molecule Cy3 via click chemistry. Analysis of fluorescent intensity in the altered proteins indicated varying degrees of conjugation. Additionally, phenyl resin-based RP-HPLC facilitated effective separation of modified proteins, unmodified proteins, and remaining fluorescent tags post-separation. SASA analysis provided insights into conjugation trends, guiding the identification of proteins amenable to LplA's tag-free modification. Our findings demonstrate LplA's broad substrate tolerability for protein modification.

Asunto(s)

Proteínas de Escherichia coli , Especificidad por Sustrato , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Química Clic , Colorantes Fluorescentes/química , Ligasas

RESUMEN

Allosteric feedback inhibition of the committed step in amino acid biosynthetic pathways is a major concern for production of amino acids at industrial scale. Anthranilate synthase (AS) catalyzes the first reaction of tryptophan biosynthetic pathway found in microorganisms and is feedback inhibited by its own product i.e. tryptophan. Here, we identified new mutant sites in AS using computational mutagenesis approach. MD simulations (20 ns) followed by MMPBSA and per residue decomposition energy analysis identified seven amino acid residues with best binding affinity for tryptophan. All 19 mutant structures were generated for each identified amino acid residue followed by simulation to evaluate effect of mutation on protein stability. Later, molecular docking studies were employed to generate mutant-tryptophan complex and structures with binding energies (kcal/mol) much higher than wild-type AS were selected. Finally, two mutants i.e., S37W and S37H were identified on the basis of positive binding scores and loss of tryptophan binding inside pocket. Further, MD simulations run for 200 ns were performed over these mutant-tryptophan complexes followed by RMSD, RMSF, radius of gyration , solvent accessible surface area , intra-protein hydrogen bond numbers, principal component analysis, free energy landscape (FEL) and secondary structure analysis to rationale effect of mutations on stability of protein. Cross correlation analysis of mutant site amino acids (S37W) with key residues of catalytic site (G325, T326, H395 and G482) was done to evaluate the effect of mutations on catalytic site conformation. Current computational mutagenesis approach predicted two mutants S37W and S37H with proposed deregulated feedback inhibition by tryptophan and retained catalytic activity.Communicated by Ramaswamy H. Sarma.