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L,L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development.

Dobson RC, Girón I, Hudson AO - PLoS ONE (2011)

Bottom Line: Cr-DapL was dimeric in both solution and when crystallized.The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis.Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
In some bacterial species and photosynthetic cohorts, including algae, the enzyme L,L-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to L,L-diaminopimelate (L,L-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and L,L-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

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Functional complementation of the E. coli dapD/E                            mutant.Functional complementation was tested using the E. coli                                dapD/E double mutant (AOH1). The plasmids pBAD33 and                                pBAD33+Cr-DapL were selected on LB agar medium                            supplemented with 50 µg mL−1 DAP and 34 µg                                mL−1 chloramphenicol. The bacteria were grown to an                            OD of 0.5 at 600 nm, the strain were serially diluted to                                10−1, 10−2, 10−3                            and 10−4 using 0.85%                            (w/v). The strain harboring the pBAD33 and                                pBAD33+Cr-DapL was replica-plated onto LB                            medium plus 0.2% (w/v) arabinose with or                            without 50 µg mL−1 DAP. The cultures were grown                            at 30 °C for 24 hours.
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pone-0020439-g003: Functional complementation of the E. coli dapD/E mutant.Functional complementation was tested using the E. coli dapD/E double mutant (AOH1). The plasmids pBAD33 and pBAD33+Cr-DapL were selected on LB agar medium supplemented with 50 µg mL−1 DAP and 34 µg mL−1 chloramphenicol. The bacteria were grown to an OD of 0.5 at 600 nm, the strain were serially diluted to 10−1, 10−2, 10−3 and 10−4 using 0.85% (w/v). The strain harboring the pBAD33 and pBAD33+Cr-DapL was replica-plated onto LB medium plus 0.2% (w/v) arabinose with or without 50 µg mL−1 DAP. The cultures were grown at 30 °C for 24 hours.

Mentions: The E. coli mutant AOH1 is suitable for a functional complementation assay because it harbors loss-of-function mutations in dapD and dapE genes. For this strain, the cells lyse because of osmotic stress, due to the lack of meso-DAP as a cross linking amino acid in the cell wall. Thus, the strain is deemed auxotrophic for DAP. The AOH1 strain was transformed with either an empty plasmid or a plasmid expressing Cr-DapL. While the mutant is able to grow only on media supplemented with DAP, only the mutant strain expressing the algal enzyme is able to grow on DAP-free media (Figure 3). The results from this assay indicate that the enzyme is able convert THDPA to L,L-DAP directly bypassing the DapD, DapC and DapE enzymatic reactions present in the E. coli pathway (Figure 1).


L,L-diaminopimelate aminotransferase from Chlamydomonas reinhardtii: a target for algaecide development.

Dobson RC, Girón I, Hudson AO - PLoS ONE (2011)

Functional complementation of the E. coli dapD/E                            mutant.Functional complementation was tested using the E. coli                                dapD/E double mutant (AOH1). The plasmids pBAD33 and                                pBAD33+Cr-DapL were selected on LB agar medium                            supplemented with 50 µg mL−1 DAP and 34 µg                                mL−1 chloramphenicol. The bacteria were grown to an                            OD of 0.5 at 600 nm, the strain were serially diluted to                                10−1, 10−2, 10−3                            and 10−4 using 0.85%                            (w/v). The strain harboring the pBAD33 and                                pBAD33+Cr-DapL was replica-plated onto LB                            medium plus 0.2% (w/v) arabinose with or                            without 50 µg mL−1 DAP. The cultures were grown                            at 30 °C for 24 hours.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3102117&req=5

pone-0020439-g003: Functional complementation of the E. coli dapD/E mutant.Functional complementation was tested using the E. coli dapD/E double mutant (AOH1). The plasmids pBAD33 and pBAD33+Cr-DapL were selected on LB agar medium supplemented with 50 µg mL−1 DAP and 34 µg mL−1 chloramphenicol. The bacteria were grown to an OD of 0.5 at 600 nm, the strain were serially diluted to 10−1, 10−2, 10−3 and 10−4 using 0.85% (w/v). The strain harboring the pBAD33 and pBAD33+Cr-DapL was replica-plated onto LB medium plus 0.2% (w/v) arabinose with or without 50 µg mL−1 DAP. The cultures were grown at 30 °C for 24 hours.
Mentions: The E. coli mutant AOH1 is suitable for a functional complementation assay because it harbors loss-of-function mutations in dapD and dapE genes. For this strain, the cells lyse because of osmotic stress, due to the lack of meso-DAP as a cross linking amino acid in the cell wall. Thus, the strain is deemed auxotrophic for DAP. The AOH1 strain was transformed with either an empty plasmid or a plasmid expressing Cr-DapL. While the mutant is able to grow only on media supplemented with DAP, only the mutant strain expressing the algal enzyme is able to grow on DAP-free media (Figure 3). The results from this assay indicate that the enzyme is able convert THDPA to L,L-DAP directly bypassing the DapD, DapC and DapE enzymatic reactions present in the E. coli pathway (Figure 1).

Bottom Line: Cr-DapL was dimeric in both solution and when crystallized.The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis.Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Victoria, Australia.

ABSTRACT
In some bacterial species and photosynthetic cohorts, including algae, the enzyme L,L-diaminopimelate aminotransferase (DapL) (E.C. 2.6.1.83) is involved in the anabolism of the essential amino acid L-lysine. DapL catalyzes the conversion of tetrahydrodipicolinate (THDPA) to L,L-diaminopimelate (L,L-DAP), in one step bypassing the DapD, DapC and DapE enzymatic reactions present in the acyl DAP pathways. Here we present an in vivo and in vitro characterization of the DapL ortholog from the alga Chlamydomonas reinhardtii (Cr-DapL). The in vivo analysis illustrated that the enzyme is able to functionally complement the E. coli dap auxotrophs and was essential for plant development in Arabidopsis. In vitro, the enzyme was able to inter-convert THDPA and L,L-DAP, showing strong substrate specificity. Cr-DapL was dimeric in both solution and when crystallized. The structure of Cr-DapL was solved in its apo form, showing an overall architecture of a α/β protein with each monomer in the dimer adopting a pyridoxal phosphate-dependent transferase-like fold in a V-shaped conformation. The active site comprises residues from both monomers in the dimer and shows some rearrangement when compared to the apo-DapL structure from Arabidopsis. Since animals do not possess the enzymatic machinery necessary for the de novo synthesis of the amino acid L-lysine, enzymes involved in this pathway are attractive targets for the development of antibiotics, herbicides and algaecides.

Show MeSH
Related in: MedlinePlus