title: Identification of the bona fide DHDPS from a common plant pathogen.
abstract: Agrobacterium tumefaciens is a Gram-negative soil-borne bacterium that causes Crown Gall disease in many economically important crops. The absence of a suitable chemical treatment means there is a need to discover new anti-Crown Gall agents and also characterize bona fide drug targets. One such target is dihydrodipicolinate synthase (DHDPS), a homo-tetrameric enzyme that catalyzes the committed step in the metabolic pathway yielding meso-diaminopimelate and lysine. Interestingly, there are 10 putative DHDPS genes annotated in the A. tumefaciens genome, including three whose structures have recently been determined (PDB IDs: 3B4U, 2HMC, and 2R8W). However, we show using quantitative enzyme kinetic assays that nine of the 10 dapA gene products, including 3B4U, 2HMC, and 2R8W, lack DHDPS function in vitro. A sequence alignment showed that the product of the dapA7 gene contains all of the conserved residues known to be important for DHDPS catalysis and allostery. This gene was cloned and the recombinant product expressed and purified. Our studies show that the purified enzyme (i) possesses DHDPS enzyme activity, (ii) is allosterically inhibited by lysine, and (iii) adopts the canonical homo-tetrameric structure in both solution and the crystal state. This study describes for the first time the structure, function and allostery of the bona fide DHDPS from A. tumefaciens, which offers insight into the rational design of pesticide agents for combating Crown Gall disease.
authors.affiliation: Department of Biochemistry, La Trobe Institute for Molecular Science, La Trobe University, Melbourne, Victoria, 3086, Australia; Department of Biochemistry and Molecular Biology, Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Victoria, 3010, Australia.
authors.forename: Sarah C
authors.initials: SC
authors.lastname: Atkinson
authors.forename: Lilian
authors.initials: L
authors.lastname: Hor
authors.forename: Con
authors.initials: C
authors.lastname: Dogovski
authors.forename: Renwick C J
authors.initials: RC
authors.lastname: Dobson
authors.forename: Matthew A
authors.initials: MA
authors.lastname: Perugini
mesh_terms: D016960:Agrobacterium tumefaciens / Q000201:enzymology* / Q000235:genetics; D000595:Amino Acid Sequence; D001483:Base Sequence; D020134:Catalytic Domain; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D006836:Hydro-Lyases / Q000096:biosynthesis / Q000235:genetics / Q000648:ultrastructure*; D010941:Plant Tumors / Q000382:microbiology; D017433:Protein Structure, Secondary; D016415:Sequence Alignment; D017422:Sequence Analysis, DNA
chemical_list: D006836:Hydro-Lyases; C020040:4-hydroxy-tetrahydrodipicolinate synthase
keywords: Crown Gall disease; X-ray crystallography; allostery; analytical ultracentrifugation; dihydrodipicolinate synthase; enzyme kinetics; enzyme structure; lysine biosynthesis