Phenol/cresol degradation by the thermophilic Bacillus thermoglucosidasiusA7: cloning and sequence analysis of five genes involved in the pathway

Bacillus thermoglucosidasius A7 degraded phenol at 65 degreesC via the meta cleavage pathway. Five enzymes used in the metabolism of phenol were clone d from B. thermoglucosidasius A7 into pUC18. Nine open reading frames were present on the 8.1 kb insert, six of which could be assigned a function in phenol degradation using database homologies and enzyme activities. The phe nol hydroxylase is a two-component enzyme encoded by pheA1 and pheA2. The l arger component (50 kDa) has 49% amino acid identity with the 4-hydroxyphen ylacetate hydroxylase of Escherichia coli, while the smaller component (19 kDa) is most related (30% amino acid identity) to the styrene monoxygenase component B from Pseudomonas fluorescens. Both components were neccessary f or activity. The catechol 2,3-dioxygenase encoded by pheB has 45% amino aci d identity with dmpB of Pseudomonas sp. CF600 and could be assigned to supe rfamily I, family 2 and a new subfamily of the Eltis and Bolin grouping. Th e 2-hydroxymuconic acid semialdehyde hydrolase (2HMSH), encoded by pheC, re vealed the highest amino acid identity (36%) to the equivalent enzyme from Pseudomonas sp. strain CF600, encoded by dmpD. Based on sequence identity, pheD and pheE were deduced to encode the 2-hydroxypenta-2,4-dienoate hydrat ase (2HDH), demonstrating 45% amino acid identity to the gene product of cu mE from Pseudomonas fluorescens and the acetaldehyde dehydrogenase (acylati ng) demonstrating 57% amino acid identity to the gene product of bphJ from Pseudomonas LB400. (C) 2000 Elsevier Science B.V. All rights reserved.