Authors:

Cait Moffatt

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The LpxC enzyme is essential in the synthesis of lipopolysaccharide, a key component of the outer membrane in gram negative bacteria, like Escherichia coli, making it an excellent target for novel antibiotics. This treatment potential has inspired many people to make mutants of the protein, targeting key amino acid sites. Although many mutations have been introduced, many of them have not been visualized within the context of the cell. We mutated H265, an amino acid important in the catalytic activity of the enzyme, into an alanine, which may later lead to an inability for the cell to form the outer membrane. We designed PCR primers to amplify a plasmid containing the LpxC gene and the Lac operon. In the primers, we replaced the 5'-CAT-3' codon with 5'-GCG-3' to introduce the H265A mutation in E. coli. After our mutant plasmids were amplified, we transformed E. coli cells to uptake them. We then ran protein expression tests, using varying amounts of IPTG to produce different amounts of wild-type LpxC to determine optimal protein expression conditions for when we purify our mutants. We ran our purified proteins on an SDS-PAGE gel. We plan to do the same approach with the mutant LpxC plasmids and overexpress them to purify the mutant protein. We will then visualize both the mutant and wildtype cells to see how the outer membrane compares between the two. If the bacterial cells cannot survive with the mutation, it could provide valuable information for the development of novel antibiotics. Exploring the Impact of Amino Acid Substitution F192D in the Deacetylase LpxC on Membrane Biosynthesis in Escherichia coli Ronni Fleming, Cait Moffatt, Sien Verschave, Daniel Kahne Harvard College | Leverett House | Human Developmental and Regenerative Biology | 2028 Lipid A is a key component of lipopolysaccharide (LPS), a glycolipid residing on the outer membrane of Gram-negative bacteria like Escherichia coli (E. coli) that confers antibiotic resistance. Our study focuses on the enzyme LpxC, which initiates production of Lipid A by deacetylating a precursor molecule, myr- UDP-GlcNAc, to form myr-UDP-GlcN. Since LpxC inhibition would prevent LPS formation, this enzyme has been identified as a promising target of antibiotics, but more research is needed on the specific nature of enzyme-substrate interactions at various positions. We are testing the impact of a two-nucleotide substitution in the coding sequence that changes residue 192 of LpxC from a phenylalanine to an aspartic acid (F192D). We introduced the F192D mutation using site-directed mutagenesis into a plasmid containing LpxC. After transforming E. coli cells to contain and express our genetically modified LpxC, we aim to compare its efficacy in producing the Lipid A precursor molecule to that of wild-type LpxC by examining cell envelope composition via electron microscopy and confirming adequate protein production using affinity chromatography. Former research revealed a 700-fold decrease in catalytic efficiency of LpxC following a substitution of Phe-192 with the smaller Ala-192, but an alteration to Asp differs because it introduces a negative charge to a formerly nonpolar position. In the wild type enzyme, weak Van der Waals interactions form between the substrate's glucosamine group and LpxC side chains (including Phe-192) in this area; we hypothesize that the charged amino acid will cause stronger polar bonds to replace them. The alteration may increase Lipid A production by promoting substrate binding or decrease it by impeding product release. The impact of our engineered F192D LpxC mutant on ultimate LPS formation will enhance our understanding of the interactions between LpxC and its substrate at this residue, helping inform future drug design targeting Gram-negative species. Harvard Summer Undergraduate Research Village

Source:

Harvard / Neuroscience / 2028

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Co-authors:

Cait Moffatt