6rg0
Structure of pdxj
Structural highlights
FunctionPDXJ_ECOLI Catalyzes the complicated ring closure reaction between the two acyclic compounds 1-deoxy-D-xylulose-5-phosphate (DXP) and 3-amino-2-oxopropyl phosphate (1-amino-acetone-3-phosphate or AAP) to form pyridoxine 5'-phosphate (PNP) and inorganic phosphate.[1] Publication Abstract from PubMedThe potential of the frequently encountered (betaalpha)8-barrel fold to acquire new functions was tested by an approach combining random mutagenesis and selection in vivo. For this purpose, the genes encoding 52 different phosphate-binding (betaalpha)8-barrel proteins were subjected to error-prone PCR and cloned into an expression plasmid. The resulting mixed repertoire was used to transform different auxotrophic Escherichia 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 mutants of the E. coli genes nanE (encoding a putative N-acetylmannosamine-6-phosphate 2-epimerase) and pdxJ (encoding the pyridoxine 5'-phosphate synthase) were responsible for rescuing both DeltaserB and DeltaserC. Unexpectedly, the complementing NanE and PdxJ variants did not catalyze the SerB or SerC reactions in vitro. Instead, 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 indicate that HisC is promiscuous for the SerC reaction, as well. The successful rescue of DeltaserB and DeltaserC through point mutations and recruitment of additional amino acids in NanE and PdxJ provides another example for the adaptability of the (betaalpha)8-barrel fold. Library Selection with a Randomized Repertoire of (betaalpha)8-Barrel Enzymes Results in Unexpected Induction of Gene Expression.,Rohweder B, Lehmann G, Eichner N, Polen T, Rajendran C, Ruperti F, Linde M, Treiber T, Jung O, Dettmer K, Meister G, Bott M, Gronwald W, Sterner R Biochemistry. 2019 Oct 15;58(41):4207-4217. doi: 10.1021/acs.biochem.9b00579., Epub 2019 Oct 7. PMID:31557000[2] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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