The DNA region encoding biphenyl dioxygenase, the first enzyme in the biphenyl-polychlorinated biphenyl degradation pathway of Pseudomonas species strain LB400, was sequenced. Six open reading frames were identified, four of which are homologous to the components of toluene dioxygenase from Pseudomonas putida Fl and have been named bphA, bphE, bphF, and bphG. From this comparison, biphenyl dioxygenase was found to be a multicomponent enzyme containing a two-subunit iron-sulfur protein, a ferredoxin, and a reductase.Comparison of the large subunit of the iron-sulfur protein and the ferredoxin with other multicomponent dioxygenases identified amino acid sequences similar to Rieske iron-sulfur proteins for binding a cluster. Sequences have also been identified in the reductase component that match the consensus sequence for FAD or NAD binding. Transcription of the biphenyl dioxygenase region was examined, and three transcription initiation sites were identified. Transcription initiating at the site furthest upstream is greatly increased when the LB400 cells are grown on biphenyl as the sole carbon source.

The polychlorinated biphenyl (PCB) congener specificities and partial BphA sequences of biphenyl dioxygenase were determined for a set of PCB-degrading bacteria. The strains examined were categorized into two groups based on their ability to degrade 17 PCB congeners. Strains that degraded a broad range of PCBs but had relatively weak activity against di-para-substituted PCBs were designated as having an LB400-type specificity. Strains designated as having a KF707-type specificity degraded a much narrower range of PCBs but had strong activity against certain di-para-substituted congeners. BphA protein sequence comparisons between these two types of strains identified four regions (designated I, II, III, and IV) in which specific sequences were consistently associated with either broad or narrow PCB substrate specificity. The dramatic differences in substrate specificity between LB400 and KF707 appear to result primarily from a combination of mutations in regions III and IV. Altering these regions in the LB400 BphA subunit to correspond to those in the KF707 sequence produced a narrow substrate specificity very similar to that of KF707. Some individual mutations within region III alone were found to improve PCB degradative activity, especially for di-para-substituted congeners. However, the greatest improvements in activity resulted from multiple amino acid modifications in region III, suggesting that the effects of these mutations are cooperative. These results demonstrate the ability to significantly improve PCB oxidative activity through sequence modifications of biphenyl dioxygenase.

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Biphenyl dioxygenase catalyzes the first step in the aerobic degradation of polychlorinated biphenyls (PCBs). The nucleotide and amino acid sequences of the biphenyl dioxygenases from two PCB-degrading strains (Pseudomonas sp. strain LB400 and Pseudomonas pseudoakaligenes KF707) were compared. The sequences were found to be nearly identical, yet these enzymes exhibited dramatically different substrate specificities for PCBs. Site-directed mutagenesis of the LB400 bphA gene resulted in an enzyme combining the broad congener specificity of LB400 with increased activity against several congeners characteristic of KF707. These data strongly suggest that the BphA subunit of biphenyl dioxygenase plays an important role in determining substrate selectivity. Further alteration of this enzyme can be used to develop a greater understanding of the structural basis for congener specificity and to broaden the range of degradable PCB congeners. Polychlorinated biphenyls (PCBs) are a group of synthetic compounds composed of biphenyl molecules containing from 1 to 10 chlorines. The vast majority of PCBs in the environment are derived from commercial mixtures (e.g., Aroclors) which contain 60 to 80 different congeners (20). Bacteria able to aerobically degrade PCBs are relatively common; however, in most cases these organisms have narrow congener specificity and are able to degrade only a small number of lightly chlorinated PCBs

This article is available online at http://www.jlr.org endoplasmic reticulum (ER) lumenal lipid droplets ( 10-12 ). These lipid droplets are believed to serve as lipid donors for bulk lipidation of primordial apoB-containing particles ( 10-13 ). One enzyme that has been implicated to play a role in the hydrolysis of stored TG pools and assembly of VLDL is an ER-associated triacylglycerol hydrolase (TGH) (14)(15)(16)(17)(18)(19)(20). Inhibition of TGH leads to decreased TG mobilization and apoB secretion from hepatocytes ( 15 ). However, treatment of hepatocytes with a TGH-specifi c inhibitor reduced secretion of TG and apoB to a lesser extent than the general lipase inhibitor, diethyl p -nitrophenyl phosphate (E600), suggesting that other lipases may also contribute to VLDL assembly ( 15 ). The fact that perinatal rat hepatocytes are capable of TG secretion in the absence of TGH expression in this particular developmental stage provides further support for the existence of additional lipases ( 21,22 ).In addition to VLDL assembly, fatty acids released from intracellular stores can be utilized for energy production via ␤ -oxidation in the mitochondria. Unlike the well-described roles of adipose triglyceride lipase (ATGL) and hormonesensitive lipase (HSL) in the mobilization of TG stores in adipose tissue ( 23-26 ), the identity of hepatic lipases supporting ␤ -oxidation is still unclear. Arylacetamide deacetylase (AADA) shares sequence homology with HSL ( 27 ) and possesses a classical lipase/esterase GXSXG active site motif ( 27,28 ). Trickett et al. ( 28 ) have shown that hepatic AADA mRNA levels follow a diurnal rhythm with an identical pattern to hepatic VLDL secretion in mice. In this study, we Hydrolysis of hepatic intracellular triacylglycerol (TG) stores generates substrates for ␤ -oxidation and lipid resynthesis, some of which are utilized for the assembly of VLDLs ( 1-3 ). The assembly of VLDL involves both cotranslational and posttranslational addition of lipids to apolipoprotein B (apoB) ( 4-7 ). The transfer of lipids to nascent apoB particles is facilitated by the microsomal triglyceride transfer protein ( 8, 9 ). Microsomal triglyceride transfer protein is also responsible for the formation of apoB-free This work was supported by a Grant-in-Aid from the Heart and Stroke Foundation of Alberta, Northwest Territories, and Nunavut. V.L., B.E., and M.T.-H.