Dataset Information

Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens.

ABSTRACT: Class A penicillin-binding proteins (PBPs) are active in the final step of bacterial peptidoglycan biosynthesis. They possess a transglycosylase (TG) domain to polymerize the glycan chains and a transpeptidase (TP) domain to catalyze peptide cross-linking. We reported that knockout of the Pbp gene in the moss Physcomitrella patens (?PpPbp) results in a macrochloroplast phenotype by affecting plastid division. Here, expression of PpPBP-GFP in ?PpPbp restored the wild-type phenotype and GFP fluorescence was observed mainly in the periphery of each chloroplast. Stable transformants expressing Anabaena PBP with the plastid-targeting sequence, or PpPBP replacing the Anabaena TP domain exhibited partial recovery, while chloroplast number was recovered to that of wild-type plants in the transformant expressing PpPBP replacing the Anabaena TG domain. Transient expression experiments with site-directed mutagenized PpPBP showed that mutations in the conserved amino acids in both domains interfered with phenotype recovery. These results suggest that both TG and TP functions are essential for function of PpPBP in moss chloroplast division.

SUBMITTER: Takahashi Y

PROVIDER: S-EPMC5328786 | biostudies-literature | 2016

REPOSITORIES: biostudies-literature

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Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens.

Takahashi Yoshiko Y Takechi Katsuaki K Takio Susumu S Takano Hiroyoshi H

Proceedings of the Japan Academy. Series B, Physical and biological sciences 20160101 10

Class A penicillin-binding proteins (PBPs) are active in the final step of bacterial peptidoglycan biosynthesis. They possess a transglycosylase (TG) domain to polymerize the glycan chains and a transpeptidase (TP) domain to catalyze peptide cross-linking. We reported that knockout of the Pbp gene in the moss Physcomitrella patens (ΔPpPbp) results in a macrochloroplast phenotype by affecting plastid division. Here, expression of PpPBP-GFP in ΔPpPbp restored the wild-type phenotype and GFP fluore ...[more]

PMID: 27941308

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Project description:Bacterial cell division is a complex process requiring the coordination of multiple components to allow the appropriate spatial and temporal control of septum formation and cell scission. Peptidoglycan (PG) is the major structural component of the septum, and our recent studies in the human pathogen Staphylococcus aureus have revealed a complex, multistage PG architecture that develops during septation. Penicillin-binding proteins (PBPs) are essential for the final steps of PG biosynthesis; their transpeptidase activity links the peptide side chains of nascent glycan strands. PBP1 is required for cell division in S. aureus, and here, we demonstrate that it has multiple essential functions associated with its enzymatic activity and as a regulator of division. Loss of PBP1, or just its C-terminal PASTA domains, results in cessation of division at the point of septal plate formation. The PASTA domains can bind PG and thereby potentially coordinate the cell division process. The transpeptidase activity of PBP1 is also essential, but its loss leads to a strikingly different phenotype of thickened and aberrant septa, which is phenocopied by the morphological effects of adding the PBP1-specific β-lactam, meropenem. Together, these results lead to a model for septal PG synthesis where PBP1 enzyme activity is required for the characteristic architecture of the septum and PBP1 protein molecules enable the formation of the septal plate. IMPORTANCE Bacterial cell wall peptidoglycan is essential, and its synthesis is the target of clinically important antibiotics such as β-lactams. β-lactams target penicillin-binding proteins (PBPs) that assemble new peptidoglycan from its building blocks. The human pathogen Staphylococcus aureus only has two essential PBPs that can carry out all the functions necessary for growth and division. In the absence of the confounding antibiotic resistance-associated PBP PBP2A, PBP1 is required for cell division, and here, we have found that it has several essential functions, both as an enzyme and as a coordinator by binding to cell division proteins and to its peptidoglycan product, via its PASTA domains. This has led to a new model for cell division with PBP1 responsible for the synthesis of the characteristic architectural features of the septum.

Project description:BackgroundThe moss Physcomitrella patens contains C18- as well as C20-polyunsaturated fatty acids that can be metabolized by different enzymes to form oxylipins such as the cyclopentenone cis(+)-12-oxo phytodienoic acid. Mutants defective in the biosynthesis of cyclopentenones showed reduced fertility, aberrant sporophyte morphology and interrupted sporogenesis. The initial step in this biosynthetic route is the conversion of a fatty acid hydroperoxide to an allene oxide. This reaction is catalyzed by allene oxide synthase (AOS) belonging as hydroperoxide lyase (HPL) to the cytochrome P450 family Cyp74. In this study we characterized two AOS from P. patens, PpAOS1 and PpAOS2.ResultsOur results show that PpAOS1 is highly active with both C18 and C20-hydroperoxy-fatty acid substrates, whereas PpAOS2 is fully active only with C20-substrates, exhibiting trace activity (~1000-fold lower kcat/KM) with C18 substrates. Analysis of products of PpAOS1 and PpHPL further demonstrated that both enzymes have an inherent side activity mirroring the close inter-connection of AOS and HPL catalysis. By employing site directed mutagenesis we provide evidence that single amino acid residues in the active site are also determining the catalytic activity of a 9-/13-AOS - a finding that previously has only been reported for substrate specific 13-AOS. However, PpHPL cannot be converted into an AOS by exchanging the same determinant. Localization studies using YFP-labeled AOS showed that PpAOS2 is localized in the plastid while PpAOS1 may be found in the cytosol. Analysis of the wound-induced cis(+)-12-oxo phytodienoic acid accumulation in PpAOS1 and PpAOS2 single knock-out mutants showed that disruption of PpAOS1, in contrast to PpAOS2, results in a significantly decreased cis(+)-12-oxo phytodienoic acid formation. However, the knock-out mutants of neither PpAOS1 nor PpAOS2 showed reduced fertility, aberrant sporophyte morphology or interrupted sporogenesis.ConclusionsOur study highlights five findings regarding the oxylipin metabolism in P. patens: (i) Both AOS isoforms are capable of metabolizing C18- and C20-derived substrates with different specificities suggesting that both enzymes might have different functions. (ii) Site directed mutagenesis demonstrated that the catalytic trajectories of 9-/13-PpAOS1 and PpHPL are closely inter-connected and PpAOS1 can be inter-converted by a single amino acid exchange into a HPL. (iii) In contrast to PpAOS1, PpAOS2 is localized in the plastid where oxylipin metabolism takes place. (iv) PpAOS1 is essential for wound-induced accumulation of cis(+)-12-oxo phytodienoic acid while PpAOS2 appears not to be involved in the process. (v) Knock-out mutants of neither AOS showed a deviating morphological phenotype suggesting that there are overlapping functions with other Cyp74 enzymes.

Dataset Information

Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens.

Publications

Both the transglycosylase and transpeptidase functions in plastid penicillin-binding protein are essential for plastid division in Physcomitrella patens.

Similar Datasets

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