MM, kcat/Km = 0.4 104 M-1 -1) (SI Appendix, Fig. S9). We note that DhpH exhibited transamination activity with unphosphorylated compounds such as SerP and L-Ala(P) in the presence of pyruvate as amino acceptor (SI Appendix, Fig. S10). In summary, the in vitro studies on the PLP-dependent enzymes DhpD and DhpH-N did not provide confirmation of the proposed pathway in Fig. 1 E and F, but DhpH generated AP from pSer(P) and DhpD converted AP to L-Ala(P) (Fig. 3A), prompting further evaluation whether these reactions could be consecutive steps in the DHP biosynthetic pathway.In Vitro Reconstitution of DhpH tRNA-Dependent Activity. A PSIBLAST search using the last 346 amino acids of DhpH as query against the UniProtKB database at the PredictProtein server (24) revealed that this part of the protein shares similarity with numerous uncharacterized proteins that belong to the domain of unknown function 482 (25). The latter is a member of the acetyltransferase clan, which consist of 30 families including the Fem resistance family members FemXAB (26, 27) and the leucyl/phenylalanyltRNA protein transferase family (28). Indeed, a BLAST search of the Protein Data Bank database (29) pinpointed FemX from Weissella viridescens (FemXWv) as the closest 3D structural match to the C-terminal domain of DhpH (sequence identity: 17 ; similarity: 29 ). When we purified the full-length DhpH protein, we observed a high A260/A280 ratio (approximately 1.3) after IMAC and desalting purification steps, a strong indicator of nucleic acid copurification. We also cloned and overexpressed in E. coli the Cterminal domain of DhpH as a histidine-tagged fusion protein (His6-DhpH-C) and observed the same high A260/A280 ratio. In contrast, purified His6-DhpH-N exhibited a A260/A280 ratio of 0.(SI Appendix, Fig. S11A). To identify the nature of the nucleic acids bound to DhpH, we treated samples of DhpH-C separately with DNase, RNase, or Proteinase K and analyzed them on a 1 agarose gel. In the RNase-treated sample, nucleic acids were no longer visible under UV light when the gel was stained with ethidium bromide, whereas nucleic acids were still visible in all other samples, suggesting that it was RNA that copurified with DhpH-C. Subsequently, we isolated the RNA by phenol extraction and ethanol precipitation. The isolated RNA sample was incubated either with L[14C(U)]-leucine in the presence of ATP and purified leucyl-tRNA synthetase from E. coli (LeuRS) or with [14C(U)]-glycine in the presence of ATP and purified glycyl-tRNA synthetase from E.SP-13786 coli (GlyRS) following a standard aminoacylation assay protocol (30).Liothyronine Only the pair L-[14C(U)]-leucine/LeuRS was able to radioactively label the RNA sample with carbon-14 (SI Appendix, Fig.PMID:24458656 S11B). To evaluate whether the C-terminal domain of DhpH could generate an amide bond, the expected product L-Leu-Ala(P) was chemically synthesized (SI Appendix, Figs. S12A and S13). This synthetic standard was first used to assess whether pSer(P) could be converted to L-Leu-Ala(P) by the coordinated action of the two domains of DhpH in the presence of Leu-tRNALeu as expected by the pathway in Fig. 1F. We set up a sensitive TLC assay in which aminoacylated tRNALeu was (re)generated in situ by LeuRS in the presence of total tRNA from E. coli, ATP, L-[14C (U)]-leucine, and thermostable inorganic pyrophosphatase (TIPP). Incubation of this regeneration system with DhpH and rac-pSer(P) did not result in formation of the desired product (Fig. 3). Thus, o.