Characterization of the Dihydroorotase from Methanococcus jannaschii

Abstract

The gene that codes for the putative dihydroorotase in the hyperthermophilic archaeon Methanococcus jannaschii was subcloned in pET-21a and expressed in Escherichia coli. A purification protocol was devised. The purity of the protein was evaluated by SDS-PAGE and the protein was confirmed by sequencing using LC-MS. The calculated molecular mass is 48104 Da. SEC-LS suggested that the protein is a monomer in solution. ICP-MS showed that there are two Zn ions per monomer. Kinetic analysis of the recombinant protein gave hyperbolic kinetics with Vmax = 12.2 µmol min−1 mg−1 and Km = 0.14 mM at 25° C. Furthermore the activity of the protein increased with temperature consistent with the hyperthermophilic nature of the organism. A homology model was constructed using the mesophilic Bacillus anthracis protein as the template. Residues known to be critical for Zn and substrate binding were conserved. The activity of the enzyme at 85° C and 90° C was found to be relatively constant over 160 min and this correlates with the temperature of optimal growth of the organism of 85° C. The amino acid sequences and structures of the two proteins were compared and this gave insight into some of the factors that may confer thermostability – more Lys and Ile, fewer Ala, Thr, Gln and Gly residues, and shorter N- and C-termini. Additional and better insight into the thermostabilization strategies adopted by this enzyme will be provided when its crystal structure is determined.

Screening and Molecular Docking of Novel Benzothiazole Derivatives as Potential Antimicrobial Agents

“…In addition, dihydroorotase was found to be essential for the survival of S. aureus [42]. Similar findings were also introduced in Gram-negative bacteria by others [40]. Moreover, this enzyme is equally essential in fungal cell metabolism [43].…”

“…The crystal structure of the dihydroorotase from E. coli was determined and it was found that the active site contains two zinc ions, which are bridged by a hydroxide group or a water molecule [39]. Structural studies confirmed the prediction that dihydroorotase is a member of the amidohydrolase superfamily with a (βα)8-barrel protein fold [39,40]. Several publications have investigated the role of inhibition of dihydroorotase in both Gram-positive and Gram-negative bacteria, which indicated its ubiquitous distribution and applicability as a suitable target for antimicrobial drug discovery.…”

Enzyme Kinetics of Recombinant Dihydroorotase from Methanococcus jannaschii

“…The protein used was the same as in the earlier study and the preparation procedure was described in that paper (Vitali et al 2017). The dihydroorotase activity in the biosynthetic direction was studied in 100 mM Mes pH 5.8 by measuring the dihydroorotate formed per minute at 230 nm, ε230 nm = 1.17 mM -1 cm -1 (Washabaugh and Collins 1984) using substrate concentrations to 9.08 mM.…”

“…A previous physicochemical characterization of Mj DHOase gave insight into its similarities and differences from the DHOases of the other types and subtypes (Vitali et al 2017). Mj DHOase is a monomer-unlike most DHOases that form dimers-and is most similar to the bacterial type I DHOase from Bacillus anthracis (Ba DHOase).…”

“…It contains 2 Zn ions in the active site unlike Aa DHOase that has only one. Previously, the kinetic behavior of Mj DHOase was studied only in the degradative direction as typically, at physiological pH, the degradative reaction is favored (Vitali et al 2017). The adaptation of the enzyme at high temperatures was also discussed.…”