Microorganisms able to grow in extreme conditions, including high concentration of salts, alkaline pH, low or high temperature and organic solvent medium have been an important source of robust enzymes for various practical applications [1]. Haloalkane dehalogenases (HLDs; EC 3.8.1.5) are predominantly bacterial enzymes that catalyze hydrolytic cleavage of a carbon-halogen bond in a broad range of halogenated aliphatic compounds, producing a corresponding alcohol, a halide anion and a proton [2]. These enzymes can be use in bioremediation, decontamination of warfare agents, synthesis of optically pure compounds, biosensing and cell imaging [3].
A novel member of HLD family, DsvA from Saccharomonospora
viridis DSM 43017 isolated from peatbogs of Ireland, has been subjected to
detailed biochemical characterization in this study. The enzyme was
successfully expressed in Escherichia coli BL21(DE3) cells and purified
to homogeneity by metalloaffinity chromatography. Proper folding and
thermostability was assessed by circular dichroism spectroscopy. Although DsvA
exhibited comparable melting temperature (Tm = 58 °C)
with other HLDs isolated from mesophilic organisms, its kinetic stability
determined at 45 and 60 °C was significantly higher than the
kinetic stability of other HLDs. Interestingly, DsvA possesses only one,
instead of two, halide-stabilizing residues in its active site previously
observed in majority of characterized HLDs. Despite unusual composition of
catalytic residues, DsvA exhibited clear dehalogenase activity. The highest
activity of the enzyme was determined towards
1-bromoheptane, 1-iodohexane and1-bromohexane, whereas no activity was detected
in the reaction with bulky and cyclic chlorinated substrates. The temperature
and pH optima of DsvA were measured with 1-iodohexane. Maximal activity was
detected between 45 and 50 °C and at pH 8.9. Steady state kinetic analyses
were performed with 1-iodohexane, 1,2-dibromoethane, 1-iodohexane and
1,3-dibromopropane. The complex kinetic mechanism of the enzyme with
1-iodohexane was determined and revealed substrate inhibition. The kinetics of
DsvA with 1,3-dibromopropane followed a simple Michelis-Menten dependence,
while the kinetics of the enzyme with 1,2-dibromoethane followed a mechanism
involving positive cooperative substrate binding. Crystallization of DsvA for
structural analysis is currently in progress.