Biphenyl dioxygenases: functional versatilities and directed evolution.

Biphenyl is a compound in which two benzene rings are connected to each other. Polychlorinated biphenyls (PCBs) can be produced by the direct chlorination of biphenyl, by which 209 different compounds containing 1 to 10 chlorines can be produced. Because PCBs have been widely used for a variety of industrial purposes, these recalcitrant compounds are recognized to be some of the most serious environmental pollutants worldwide. Biphenyl-utilizing bacteria cometabolize PCBs into chlorobenzoic acids by using biphenyl-catabolic enzymes via an oxidative route (Fig. 1). Several biphenyland PCB-degrading bacteria, including both gram-negative and gram-positive strains, have been isolated to date (1, 18, 19, 81). Using these bacteria, many workers have studied the biochemical and genetic bases of PCB degradation in detail. Biphenyl dioxygenase (BphA) is a Resike-type, three-component enzyme, composed of a terminal dioxygenase and an electron transfer chain (Fig. 1) (12, 49). The former consists of a large subunit and a small subunit, associating as an 3 3 heterohexamer (11, 46). The latter consists of ferredoxin and its reductase and is involved in electron transfer from NADH to reduce the terminal dioxygenase. The terminal dioxygenase activates molecular oxygen to introduce it into the biphenyl molecule at the 2,3 position to obtain a 2,3-dihydro-2,3-diol, which is then dehydrogenated to 2,3-dihydroxybiphenyl by dihydrodiol dehydrogenase (BphB). The second dioxygenase, 2,3-dihydroxybiphenyl dioxygenase (BphC), does not require any external reductant and cleaves the 2,3-dihydroxylated ring between carbon atoms 1 and 2 to produce 2-hydroxy-6-oxo-6phenylhexa-2,4-dienoic acid (HOPD, the ring meta-cleavage product), which is then hydrolyzed to benzoic acid and 2-hydroxypenta-2,4-dienoate by a hydrolase (BphD). These upper pathway enzymes in biphenyl metabolism are encoded by the bph gene clusters, in which bphA1 and bphA2 encode a large and a small subunit of the terminal dioxygenase, bphA3 encodes ferredoxin, and bphA4 encodes ferredoxin reductase (Fig. 1) (15, 20, 28, 36, 48, 79). The bphB, bphC, and bphD genes encode a dehydrogenase, a ring-cleavage dioxygenase, and a hydrolase, respectively. Among these, the large subunit of terminal dioxygenase is crucially involved in the substrate specificity of biphenyl dioxygenase (40, 42). Therefore, evolutionary molecular engineering has been applied to large-subunit genes of different origins, creating novel dioxygenases. Evolved biphenyl dioxygenases thus obtained show enhanced and expanded degradation for not only PCBs, but also other related compounds (7, 8, 40, 42, 75–77). The use of evolved enzymes is also effective for the synthesis of high-value organic molecules in the pharmaceutical industries (53, 72). In this communication, we review recent advances in studies on the function, regulation, and engineering of bph genes, particularly focusing on the versatile characteristics of biphenyl dioxygenases.