Characterization and Evolutionary Implications of the Triad Asp-Xxx-Glu in Group II Phosphopantetheinyl Transferases

Abstract

Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.

Two Bacterial Group II Phosphopantetheinyl Transferases Involved in Both Primary Metabolism and Secondary Metabolism

“…E. coli BL21(DE3) was used for proteins expression. Protein sequences of the Hppt and Sppt were obtained from the National Center for Biotechnology Information (NCBI) database [22].…”

“…Deletion of both His-Tag gene and Nus-Tag gene from pET44a [22] pYY0052 Hppt E cloned as a NdeI-HindIII fragment into pET44a [22] pYY0041…”

“…Hppt E and sppt E were chemically synthesized and cloned as NdeI/HindIII fragments into pET44a, yielding plasmids pYY0052 and pYY0053, respectively [22]. Regarding schPPT-sppt E , the 5 0 -DNA of schPPT was amplified by PCR using pYY0041 as template with primer pairs WYY0013/ SchPPTSppt1 [11].…”

“…The 3 0 -DNA of hppt E was amplified by PCR using pYY0052 [22] as template with primer pairs SpptHppt2/Hppt2. Then sppt E -hppt E was amplified by overlapping PCR using both 5 0 -DNA of sppt E and 3 0 -DNA of hppt E as templates with primer pairs Sppt1/ Hppt2.…”

“…Recently, it has been reported that Sppt and Hppt phosphopantetheinylate ACPs involved in primary metabolism [18,22]; PPT Ns phosphopantetheinylates ACPs and PCPs involved in secondary metabolism [4]; PcpS phosphopantetheinylates ACPs and PCPs involved in both primary metabolism and secondary metabolism [1]. In this study, we characterized the specificities of Hppt and Sppt and the effects of their N/C terminus to the activities.…”