Cloning, expression and crystallization of dihydrodipicolinate reductase from methicillin-resistant Staphylococcus aureus

Abstract

Dihydrodipicolinate reductase (DHDPR; EC 1.3.1.26) catalyzes the nucleotide (NADH/NADPH) dependent second step of the lysine-biosynthesis pathway in bacteria and plants. Here, the cloning, expression, purification, crystallization and preliminary X-ray diffraction analysis of DHDPR from methicillin-resistant Staphylococcus aureus (MRSA-DHDPR) are presented. The enzyme was crystallized in a number of forms, predominantly with ammonium sulfate as a precipitant, with the best crystal form diffracting to beyond 3.65 A resolution. Crystal structures of the apo form as well as of cofactor (NADPH) bound and inhibitor (2,6-pyridinedicarboxylate) bound forms of MRSA-DHDPR will provide insight into the structure and function of this essential enzyme and valid drug target.

Figure 1

Crystals of (a) apo MRSA-DHDPR, (b) MRSA-DHDPR in the presence of NADPH cofactor and (c) MRSA-DHDPR in the presence of NADPH and the substrate analogue 2,6-PDC.

Figure 2

(a) Agarose-gel analysis following NdeI and BamHI restriction-endonuclease digestion of the plasmid pDD003, depicting the cut pET11a vector and the dapB gene. Markers are labelled in base pairs. (b) SDS–PAGE gel showing the expression and purification of MRSA-DHDPR. Lane 1, molecular-weight markers (kDa); lane 2, E. coli BL21 (DE3) cell-free extract; lane 3, extract after induction with 1 mM IPTG for 3 h; lane 4, pooled Q-Sepharose fractions; lane 5, pooled Phenyl Sepharose fractions; lane 6, pooled Superose 12 fractions. (c) Deconvoluted ESI-TOF mass spectrum of MRSA-DHDPR showing a major peak with mass of 26 668.40 Da, which is consistent with the theoretical monomer mass of the enzyme. Inset: raw MS data plotted as intensity versus mass-to-charge ratio.

Figure 3

X-ray diffraction frames from crystals of MRSA-DHDPR co-crystallized with NADPH and 2,6-PDC.