Exploring the Impact of Amino Acid Substitution F192D in the Deacetylase LpxC on Membrane Biosynthesis in Escherichia coli

Authors:

Ronni Fleming, Cait Moffatt, Sien Verschave, Daniel Kahne

Date Created:

2025-01-01

Course Title:

Professor:

Not specified

About Paper:

Lipid A is a key component of lipopolysaccharide (LPS), a thatofwild-typeLpxCbyexaminingcellenvelopecompositionvia glycolipid residing on the outer membrane of Gram-negative electron microscopy and confirming adequate protein production bacteria like Escherichia coli (E. coli) that confers antibiotic using affinity chromatography. Former research revealed a resistance. Our study focuses on the enzyme LpxC, which initiates 700-fold decrease in catalytic efficiency of LpxC following a production of Lipid A by deacetylating a precursor molecule, myr- substitution of Phe-192 with the smaller Ala-192, but an alteration UDP-GlcNAc, to form myr-UDP-GlcN. Since LpxC inhibition to Asp differs because it introduces a negative charge to a formerly would prevent LPS formation, this enzyme has been identified nonpolar position. In the wild type enzyme, weak Van der Waals as a promising target of antibiotics, but more research is needed interactions form between the substrate’s glucosamine group and on the specific nature of enzyme-substrate interactions at various LpxC side chains (including Phe-192) in this area; we hypothesize positions. We are testing the impact of a two-nucleotide that the charged amino acid will cause stronger polar bonds to substitution in the coding sequence that changes residue 192 of replace them. The alteration may increase Lipid A production by LpxC from a phenylalanine to an aspartic acid (F192D). We promoting substrate binding or decrease it by impeding product introduced the F192D mutation using site-directed mutagenesis release. The impact of our engineered F192D LpxC mutant on into a plasmid containing LpxC. After transforming E. coli cells ultimate LPS formation will enhance our understanding of the to contain and express our genetically modified LpxC, we aim to interactions between LpxC and its substrate at this residue, helping compareitsefficacyinproducingtheLipidAprecursormoleculeto inform future drug design targeting Gram-negative species.

Abstract:

Lipid A is a key component of lipopolysaccharide (LPS), a thatofwild-typeLpxCbyexaminingcellenvelopecompositionvia glycolipid residing on the outer membrane of Gram-negative electron microscopy and confirming adequate protein production bacteria like Escherichia coli (E. coli) that confers antibiotic using affinity chromatography. Former research revealed a resistance. Our study focuses on the enzyme LpxC, which initiates 700-fold decrease in catalytic efficiency of LpxC following a production of Lipid A by deacetylating a precursor molecule, myr- substitution of Phe-192 with the smaller Ala-192, but an alteration UDP-GlcNAc, to form myr-UDP-GlcN. Since LpxC inhibition to Asp differs because it introduces a negative charge to a formerly would prevent LPS formation, this enzyme has been identified nonpolar position. In the wild type enzyme, weak Van der Waals as a promising target of antibiotics, but more research is needed interactions form between the substrate’s glucosamine group and on the specific nature of enzyme-substrate interactions at various LpxC side chains (including Phe-192) in this area; we hypothesize positions. We are testing the impact of a two-nucleotide that the charged amino acid will cause stronger polar bonds to substitution in the coding sequence that changes residue 192 of replace them. The alteration may increase Lipid A production by LpxC from a phenylalanine to an aspartic acid (F192D). We promoting substrate binding or decrease it by impeding product introduced the F192D mutation using site-directed mutagenesis release. The impact of our engineered F192D LpxC mutant on into a plasmid containing LpxC. After transforming E. coli cells ultimate LPS formation will enhance our understanding of the to contain and express our genetically modified LpxC, we aim to interactions between LpxC and its substrate at this residue, helping compareitsefficacyinproducingtheLipidAprecursormoleculeto inform future drug design targeting Gram-negative species.

Source:

Harvard / Harvard College | Eliot House | Neuroscience | 2028 / 2025

Topics:

lpxc, f192d, coli, enzyme, impact, acid, substitution, lipid, lps, production, interaction, substrate

Co-authors:

@ronnifleming192 , @caitmoffatt189 , @sienverschave190 , @danielkahne191