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Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

ABSTRACT: The (βa)8-barrel enzyme fold is frequently encountered in nature and catalyzes a multitude of different reactions. It is therefore interesting to evaluate the evolutionary potential of this fold by laboratory experiments. Along these lines, we tested the ability of the (βa)8-barrel to acquire new catalytic functions by an approach combining random mutagenesis and selection in vivo. The genes encoding 52 different phosphate-binding (βa)8-barrel proteins were randomized by error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia coli (E. coli) strains, each lacking an enzyme with a phosphate-containing substrate. After plating of the different transformants on minimal medium, growth was observed only for two strains, lacking either the gene for the serine phosphatase SerB or the phosphoserine aminotransferase SerC. The same variants of point-mutated E. coli genes nanE (encoding a putative N-acetlymannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5’-phosphate synthase) were responsible for rescuing both DserB and DserC. Remarkably, all selected PdxJ variants have acquired a 10-residue C-terminal extension from the cloning vector, that is essential for the rescue of DserB and DserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or Serc reactions in vitro. However, RT-qPCR, RNAseq, and transcriptome analysis showed that they rescue the deletions by enlisting the help of endogenous E. coli enzymes HisB and HisC through exclusive up-regulation of histidine operon transcription. While the promiscuous SerB activity of HisB is well-established, our data indicates that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DserB and DserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (βa)8-barrel fold. To our knowledge, this is the first example of (βa)8-barrels functioning not as enzymes, but by activating transcription via an unknown mechanism.

ORGANISM(S): Escherichia coli

PROVIDER: GSE133289 | GEO | 2020/02/04

REPOSITORIES: GEO

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Project description:Plasmodium falciparum is the etiological agent of human malaria, one of the most widespread diseases in tropical and subtropical world regions. One of the biggest problems in controlling the disease is the emergence of drug resistance, which leads to the need to discover new antimalarial compounds. One of the most promissory drugs purposed is fosmidomycin, an inhibitor of the biosynthesis of isoprene units by the methylerythritol 4-phosphate (MEP) pathway which in some cases failed in clinic studies. Once formed, isoprene units are condensed to form longer structures such as farnesyl and geranylgeranyl pyrophosphate (GGPP), which are necessary for heme O and A formation, ubiquinone, and dolichyl phosphate biosynthesis as well as for protein isoprenylation. Even though the natural substrates of polyprenyl transferases and syntheses are polyprenyl pyrophosphates, it was already demonstrated that isoprenoid alcohols (polyprenols) such as farnesol (FOH) and geranylgeraniol (GGOH) can rescue parasites from fosmidomycin. This study better investigated how this rescue phenomenon occurs by performing drug-rescue assays. By this, it was observed that phytol (POH), a 20-carbon plant isoprenoid, rescues parasites from the fosmidomycin effect, similarly to FOH or GGOH. Contrarily, neither dolichols nor nonaprenol rescue parasites from fosmidomycin. Considering this, here we characterized the transport of FOH, GGOH, and POH. Once incorporated, it was observed that these substances are phosphorylated, condensed into longer isoprenoid alcohols, and incorporated into proteins and dolichyl phosphates. Through proteomic and radiolabelling approaches, it was found that prenylated proteins are naturally attached to several isoprenoids including GGOH, dolichol, and POH if exogenously added. Furthermore, results suggest the presence of at least two promiscuous protein prenyltransferases in the parasite: one enzyme which can use FPP among other unidentified substrates and another enzyme that can use GGOH, POH, and dolichols among other substrates not identified here. Thus, was obtained further evidence for dolichols and other isoprenoid products attached to proteins. This study helps better understand apicoplast-targeting antimalarials mechanism of action as well as novel posttranslational modifications of proteins.

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Library selection with a randomized repertoire of (βa)8-barrel enzymes results in unexpected induction of gene expression

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