Crystal structure of dihydrodipicolinate synthase (BA3935) from <i>Bacillus anthracis</i> at 1.94 Å resolution

摘要

As part of a structural genomics program, we are determining structures of proteins from the causative agent of anthrax, Bacillus anthracis, a Gram-positive spore-forming bacterium. Among our initial candidates for crystallographic analysis are the products of essential genes based on knock-out studies in Bacillus subtilis. The BA3935 gene of B. anthracis (www.tigr.org), annotated as DapA2, encodes a putative protein consisting of 292 amino acid residues with a subunit molecular weight of 31,233 Da. The predicted protein has 60% identity with dihydrodipicolinate synthase (DHDPS or DapA) from B. subtilis and 40% amino acid sequence identity to its orthologue in Escherichia coli. dapA is one of only 271 out of the total of 4118 genes in B. subtilis that are indispensable for growth of the organism on standard laboratory media.1 DHDPS catalyses the condensation of aspartate semialdehyde and pyruvate and is the first committed step on the pathway to diaminopimelate and L-lysine in prokaryotes, some fungi, and higher plants (Scheme 1). The product released from the E. coli enzyme has been shown to be 4-hydroxy-2,3,4,5-tetrahydro-(2S)-dipicolinic acid (HTDPA) rather than L-2,3-dihydrodipicolinic acid (DHDPA); as the name of the enzyme suggests,2 DHDPA can be formed spontaneously from HTDPA by elimination of water. The steps of aspartate semialdehyde synthesis from aspartate are shared with the biosynthetic pathways leading methionine and threonine. The diaminopimelate/lysine pathway is thought to be of particular importance in Gram-positive bacteria because diaminopimelate makes up a higher proportion of the dry cell weight than it does in Gram-negative bacteria, a consequence of the thicker cell wall in the former. In Bacilli, the product of the DHDPS reaction is further reduced by dipicolinate synthase to dipicolinate, which makes up 10% of the dry weight of spores. Crystal structures have been determined of DHDPS from E. coli,3 Nicotiana sylvestris,4 and most recently from Thermotoga maritima.5 In this article, we report the crystal structure of DHDPS from B. anthracis (Ba DHDPS), the first structure of a dihydrodipicolinate synthase from a Gram-positive bacterium. The structure of Ba DHDPS was determined by molecular replacement using the coordinate set for the E. coli orthologue (PDB code 1DHP) as the search model.3 Two structures have been determined to 1.9 and 2.2 Å resolution in different orthorhombic crystal forms. Data collection, refinement, and model-building statistics are summarized in Table I. The structures in the two crystal forms are very similar and unless otherwise stated, the commentary here will refer to the structure refined to higher resolution (1XKY). Each DHDPS subunit [Fig. 1(A)] consists of a (β/α)8 barrel with three α-helices at the C-terminus of the chain. In both crystal forms, there are four subunits in the asymmetric unit and for all of these chains there is clear electron density for residues Met1 to Arg292. The chains within and between the crystal forms can be closely superimposed with pairwise root mean squared deviations (rmsΔ) in Cα coordinate positions in the range 0.24–0.34 Å. Following these superpositions, the largest deviations occur at residues 17–19 in the β1–α1 loop, 221–230 in the α8–α9 loop, and residues 290–292 at the C-terminus. When the BaDHDPS structure is compared with the EcDHDPS structure, the positional rmsΔ following least squared superposition of 290 equivalent Cα atoms is 1.35–1.45 Å. This compares with an rmsΔ of 1.45–1.55 Å for comparisons between BaDHDPS and TmDHDPS over 284 equivalent Cα atoms. Aspects of the sequence and structure of dihydrodipicolinate synthase from B. anthracis: (A) Ribbon tracing of the DHDPS subunit colour ramped from the amino- (blue) to the carboxyl-terminus (red). The side chain of the active site lysine 163 is shown in liquorice representation at the centre of the molecule. The view is looking down the axis of the β-barrel. (B) Ribbon tracing o