Cloning, expression, purification, crystallization and preliminary X-ray crystallographic study of DHNA synthetase fromGeobacillus kaustophilus

Abstract

The aerobic Gram-positive bacterium Geobacillus kaustophilus is a bacillus species that was isolated from deep-sea sediment from the Mariana Trench. 1,4-Dihydroxy-2-naphthoate (DHNA) synthetase plays a vital role in the biosynthesis of menaquinone (vitamin K(2)) in this bacterium. DHNA synthetase from Geobacillus kaustophilus was crystallized in the orthorhombic space group C222(1), with unit-cell parameters a = 77.01, b = 130.66, c = 131.69 A. The crystal diffracted to a resolution of 2.2 A. Preliminary studies and molecular-replacement calculations reveal the presence of three monomers in the asymmetric unit.

Structural Basis of the Induced-Fit Mechanism of 1,4-Dihydroxy-2-Naphthoyl Coenzyme A Synthase from the Crotonase Fold Superfamily

“…Interestingly, the C-terminal helical domain was found to cross the trimer-trimer interface to form part of the active site of the subunit in the opposite trimer. The same arrangement of the C-terminal helix is found in all the structurally known MenB enzymes, including those from Staphylococcus aureus [12], Salmonella typhimurium [13], Geobacillus kaustophilus HTA 426 [14], Escherichia coli [15], [16], and Synechocystis sp. PCC6803 [16].…”

Thermostable Mismatch-Recognizing Protein MutS Suppresses Nonspecific Amplification during Polymerase Chain Reaction (PCR)

“…The forward and reverse primers were 5′- CCATGG AGAAATCTGAGAAAGAGCTCAC-3′ and 5′ -AGATCT TTATTAAGCTCCGGACTCCTTTTT-3′, respectively, which contained NcoI and BglII sites, respectively (underlined). The amplified fragment was ligated into the NcoI and BamHI sites of pET-HisTEV [26] to yield a pET-HisTEV/ A. aeolicus mutS plasmid. Sequence analysis revealed that the construction was error free.…”

Characterization of 1,4-dihydroxy-2-naphthoyl-coenzyme A synthase (MenB) in phylloquinone biosynthesis of Synechocystis sp. PCC 6803

“…This essential role of bicarbonate is further supported by the finding of its effect on the rate of binding of the DHNA-CoA inhibitor by ScMenB, which strongly suggests that bicarbonate is bound to the ScMenB active site. Currently, except in S. typhimurium MenB, no bicarbonate is found at the active sites of available crystal structures of predicted type I DHNA-CoA synthases including E. coli MenB [37], Staphylococcus aureus MenB [40], and Geobacillus kaustophilus HTA 426 MenB (PDB entry: 2IEX) [41]. However, these crystal structures were determined in the absence of added bicarbonate.…”