Diversity of the C-Terminal Portion of the Biphenyl Dioxygenase Large Subunit

Abstract

The biphenyl dioxygenase (BPDO) catalyses a stereospecific dioxygenation of biphenyl and analogs of it. Aside from being involved in the destruction and detoxification of toxic pollutants in soil, in the context of the green chemistry concept, this enzyme is a promising biocatalyst to design new more selective and more environmentally friendly approaches to manufacture fine chemicals. At this time, most of our knowledge about the variability of key residues determining the substrate specificity and regiospecificity of the enzyme oxygenase component (BphAE) toward biphenyl analogs and about the effect of altering these residues on catalytic properties is based on investigations made with BphAEs from cultured organisms and engineered enzymes derived from them. The purpose of this work was to examine the diversity of the amino acid sequence patterns of the α subunit (BphA) C-terminal domain deduced from PCR products amplified from DNA extracted from cultured bacteria of various phylogenetic lines and from the soil microflora of PCB-contaminated soils. Of special interest were segments of the C-terminal portion called regions I, III and IV. Altogether, the phylogenetic tree obtained from aligning the deduced amino acid sequences of BphAs C-terminal domain from cultured bacteria belonging to various ecological niches and from uncultured soil bacteria reveals that most of the BphAs were linked to the three clusters of BphAs previously reported. However, few belong to new branches that diverge from the previously known branches showing a high diversity of BphAs in natural environment. Furthermore, data show a wide distribution of BphAs with family linkages that not only crosses bacterial taxonomic frontiers but also ecological niches. Nevertheless, in spite of this divergence, the sequence patterns of regions III and IV amino acids that are known to influence substrate specificity and regiospecificity are rather conserved among BphAs and the pattern was independent of the family cluster to which they belong. In most cases, regions III and IV amino acid patterns are closer to those of Pseudomonas pseudoalcaligenes KF707 BphA1 than to the most versatile Burkholderia xenovorans LB400 BphA. This might suggest that the PCB-degrading potency of soil bacteria is closer to the one observed for KF707 BphAE than from LB400 BphAE. However, the fact that among less than 20 PCR products amplified from soil DNA that we have sequenced, one of them was very homologous to that of LB400 BphA and in addition, residues 335 and 336 of LB400 were replaced by residues that previous enzyme engineering had shown to extend the range of PCB substrate used by the enzyme strongly suggest that PCB-degrading bacteria are evolving in soil to optimize their PCB-degrading capacity.

Remarkable Ability of Pandoraea pnomenusa B356 Biphenyl Dioxygenase To Metabolize Simple Flavonoids

“…strain KKS102 (not shown) which, unlike Orf0 of strain LB400, were found to be negative regulators (5,27). The phylogenetic tree obtained when sequences of known BphAs from cultured and uncultured bacteria are aligned (38) shows three branches: two comprise principally Gram-negative proteobacteria, and one comprises exclusively high-GC-content Gram-positive bacteria of the rhodococcal group. It is noteworthy that BphAE B356 clusters with BphA1A2 of strain A5 and of strain KKS102, whereas BphAE LB400 belongs to a separate branch.…”

Has the Bacterial Biphenyl Catabolic Pathway Evolved Primarily To Degrade Biphenyl? The Diphenylmethane Case

“…Biphenyl is a naturally occurring chemical, but it is not universally distributed in nature. It is noteworthy that bacteria carrying the biphenyl catabolic pathway enzymes were obtained from pristine soils not exposed to biphenyl or chlorobiphenyls (10).…”

“…On the basis of their primary amino acid sequences, BphAE B356 and BphAE LB400 belong to distinct phylogenetic clusters of BphAE (9)(10)(11). Recent data suggested that each has acquired a distinct PCB-degrading pattern (9,15), as well as distinct abilities to metabolize simple flavonoids (14), DDT (16), or DDD (43).…”

“…The catalytic center of the enzyme is located on the C-terminal portion of the BphAE ␣ subunit, which also carries the major structural determinants for substrate specificity (8). There are three phylogenetically distinct clusters of BphAEs (9)(10)(11), and the structure of a representative BphAE (also called BphA1A2) from each of these three clusters has now been elucidated. Thus, the Protein Data Bank (PDB) coordinate file for Burkholderia xenovorans LB400 BphAE (BphAE LB400 ) is available (8), as are those for Pandoraea pnomenusa B356 BphAE (BphAE B356 ) (12) and Rhodococcus jostii RHA1 BphA1A2 (BphA1A2 RHA1 ) (13).…”

Characterization of Biphenyl Dioxygenase of Pandoraea pnomenusa B-356 As a Potent Polychlorinated Biphenyl-Degrading Enzyme

“…Moreover, it preferentially transforms 4,4Ј-dichlorobiphenyl over either 3,3Ј-or 2,2Ј-dichlorobiphenyl (24). A third enzyme, BPDO B356 from Pandoraea pnomenusa (formerly Comamonas testosteroni [59]) B356, shares approximately 70% sequence identity with both BPDO KF707 and BPDO LB400 . Previous studies have indicated this enzyme's preference for meta-Ͼ ortho-Ͼ para-substituted dichlorobiphenyls (33), as well as its higher specific activity against biphenyl than that of BPDO LB00 .…”