Sequence variation in dichloromethane dehalogenases/glutathione S-transferases.

Abstract

Dichloromethane dehalogenase/glutathione S-transferase allows methylotrophic bacteria to grow with dichloromethane (DCM), a predominantly man-made compound. Bacteria growing with DCM by virtue of this enzyme have been readily isolated in the past. So far, the sequence of the dcmA gene encoding DCM dehalogenase has been determined for Methylobacterium dichloromethanicum DM4 and Methylophilus sp. DM11. DCM dehalogenase genes closely related to that of strain DM4 were amplified by PCR and cloned from total DNA from 14 different DCM-degrading strains, enrichment cultures and sludge samples from wastewater treatment plants. In total, eight different sequences encoding seven different protein sequences were obtained. Sequences of different origin were identical in several instances. Sequence variation was limited to base substitutions; strikingly, 16 of the 19 substitutions in the dcmA gene itself encoded amino acids that were different from those of the DM4 sequence. The kinetic parameters k(cat) and K:(m), the pH optimum and the stability of representative DCM dehalogenase variants were investigated, revealing minor differences between the properties of DCM dehalogenases related to that from strain DM4.