Purification, crystallization and preliminary X-ray diffraction studies to near-atomic resolution of dihydrodipicolinate synthase from methicillin-resistantStaphylococcus aureus

Abstract

In recent years, dihydrodipicolinate synthase (DHDPS; EC 4.2.1.52) has received considerable attention from both mechanistic and structural viewpoints. DHDPS is part of the diaminopimelate pathway leading to lysine, coupling (S)-aspartate--semialdehyde with pyruvate via a Schiff base to a conserved active-site lysine. In this paper, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPS from methicillin-resistant Staphylococcus aureus, an important bacterial pathogen, are reported. The enzyme was crystallized in a number of forms, predominantly from PEG precipitants, with the best crystal diffracting to beyond 1.45 Å resolution. The space group was P1 and the unit-cell parameters were a = 65.4, b = 67.6, c = 78.0 Å , = 90.1, = 68.9, = 72.3. The crystal volume per protein weight (V M ) was 2.34 Å 3 Da À1, with an estimated solvent content of 47% for four monomers per asymmetric unit. The structure of the enzyme will help to guide the design of novel therapeutics against the methicillin-resistant S. aureus pathogen.

Structural Determinants Defining the Allosteric Inhibition of an Essential Antibiotic Target

“…Cloning, Expression, and Purification of DHDPS Genomic DNA from L. pneumophila (strain Alcoy) and a clinical isolate of S. pneumoniae (serotype 3) were used in this study. The procedures for amplifying and cloning the dapA gene (encoding DHDPS) from L. pneumophila and S. pneumoniae and for the overexpression and purification of recombinant DHDPS were performed as described previously (Atkinson et al, 2011;Burgess et al, 2008b;Dogovski et al, 2013;Sibarani et al, 2010;Siddiqui et al, 2013). Briefly, dapA encoding DHDPS was PCR-amplified from genomic DNA and cloned into pET11a, pET28a, or pRSET A expression vector (Table 1).…”

“…For quantitative analysis of DHDPS enzymatic activity, this study employed a coupled assay as described previously (Atkinson et al, 2014;Burgess et al, 2008b;Dobson et al, 2005b;Dogovski et al, 2013; Voss et al, 2010). Substrate turnover was measured spectrophotometrically at A 340 nm ( 3 340 nm = 6,220 M À1 cm À1 ) via the associated oxidation of NADPH.…”

Structure and Evolution of a Novel Dimeric Enzyme from a Clinically Important Bacterial Pathogen

“…Structure Determination-X-ray diffraction data from crystals of MRSA-DHDPS were collected at the Australian Synchrotron (Clayton, Australia) and were published elsewhere (25). Diffraction data were integrated and scaled using the MOSFLM (26) and SCALA (27) software.…”

“…Solution-MRSA-DHDPS was expressed and purified as described elsewhere (25). The enzyme was initially characterized by electrospray ionization time of flight mass spectrometry and CD spectroscopy to show that the mass of the recombinant product was consistent with the amino acid sequence of the native protein after methionine cleavage (supplemental Fig.…”

Molecular Interaction of Novel Compound 2-Methylheptyl Isonicotinate Produced by Streptomyces sp. 201 with Dihydrodipicolinate Synthase (DHDPS) Enzyme of Mycobacterium tuberculosis for its Antibacterial Activity

“…The three-dimensional crystal structures of DHDPS from M. tuberculosis [13], Escherichia coli [14], Nicotiana sylvestris [15], Staphylococcus aureus [16], Bacillus anthracis [17], are available at Protein Data Bank (http:// www.rcsb.org/). The present study mainly focused on molecular docking studies of a novel compound (2-methylheptyl isonicotinate) isolated from Streptomyces sp.…”