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The maizeglossy2andglossy2-likegenes are homologs, which encode proteins that belong to the BAHD family of acyltransferases.In plantagenetic studies have demonstrated that these genes may be involved in the elongation of very long chain fatty acids (VLCFAs) that are precursors of the cuticular wax fraction of the plant cuticle. VLCFAs are synthesized by a fatty acyl-CoA elongase complex (FAE) that consists of four component enzymes. Previously, we functionally identified the maize FAE component enzymes by their ability to complement haploidSaccharomyces cerevisiaestrains that carry lethal deletion alleles for each FAE component enzyme. In this study we used these complemented haploid strains and wild-type diploid strains to evaluate whether the co-expression of either GLOSSY2 or GLOSSY2-LIKE with individual maize FAE component enzymes affects the VLCFA product-profile of the FAE system. Wild-type diploid strains produced VLCFAs of up to 28-carbon chain length. Co-expression of GLOSSY2 or GLOSSY2-LIKE with a combination of maize 3-ketoacyl-CoA synthases stimulated the synthesis of longer VLCFAs, up to 30-carbon chain lengths. However, such results could not be recapitulated when these co-expression experiments were conducted in the yeast haploid mutant strains that lacked individual components of the endogenous FAE system. Specifically, lethal yeast mutant strains that are genetically complemented by the expression of maize FAE-component enzymes produce VLCFAs that range between 20- and 26-carbon chain lengths. However, expressing either GLOSSY2 or GLOSSY2-LIKE in these complemented strains does not enable the synthesis of longer chain VLCFAs. These results indicate that the apparent stimulatory role of GLOSSY2 or GLOSSY2-LIKE to enable the synthesis of longer chain VLCFAs in diploid yeast cells may be associated with mixing plant enzyme components with the endogenous FAE complex.
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Scalable and Chromatography‐Free Synthesis of Efflux Pump Inhibitor Phenylalanine Arginine β‐Naphthylamide for Its Validation in Wild‐Type Bacterial Strains
Abstract Phenylalanine arginine β‐naphthylamine, or PAβN, is a C‐terminus capped dipeptide discovered in 1999 as an RND‐type efflux pump inhibitor (EPI). Since then, PAβN has become a standard tool compound in EPI research and development. Despite this, PAβN lacks a detailed or efficient synthesis, and standard parameters for its use in wild‐type bacterial strains are inconsistent or non‐existent. Therefore, a scalable and chromatography‐free synthesis of PAβN was developed using streamlined traditional solution‐phase peptide coupling chemistry. With this procedure, gram scale quantities of PAβN were synthesized alongside analogues and stereoisomers to build a focused library to evaluate simple structure activity relationships. While most analogues were less active than the broadly utilized L,L‐PAβN itself, we identified that its enantiomer, D,D‐PAβN, also provided 8‐ to 16‐fold potentiation of the antibiotic levofloxacin at 40 to 50 μg/mL concentrations of EPI in various wild‐typePseudomonas aeruginosastrains. Additionally, D,D‐PAβN was shown to be significantly more hydrolytically stable than L,L‐PAβN, indicating that it may be a useful, and now readily synthesized, tool compound facilitating future EPI research.
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- Award ID(s):
- 2018399
- PAR ID:
- 10419151
- Publisher / Repository:
- Wiley Blackwell (John Wiley & Sons)
- Date Published:
- Journal Name:
- ChemMedChem
- Volume:
- 18
- Issue:
- 14
- ISSN:
- 1860-7179
- Format(s):
- Medium: X
- Sponsoring Org:
- National Science Foundation
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