Rhodococcus sp. RHA1 induces two biphenyl dioxygenases, the BphA and EtbA/EbdA dioxygenases, during growth on biphenyl. Their subunit genes were expressed in R. erythropolis IAM1399 to investigate the involvement of each subunit gene in their activity and their substrate preferences. The recombinant expressing ebdA1A2A3etbA4 and that expressing bphA1A2A3A4 exhibited 4-chlorobiphenyl (4-CB) transformation activity, suggesting that these gene sets are responsible for the EtbA/EbdA and BphA dioxygenases respectively. When bphA4 and etbA4 were swapped to construct the recombinants expressing ebdA1A2A3bphA4 and bphA1A2A3etbA4 respectively, compatibility between BphA4 and EtbA4 was suggested by their 4-CB transformation activities. When bphA3 and ebdA3 were swapped, incompatibility between BphA3 and EbdA3 was suggested. BphA and EtbA/EbdA dioxygenases exhibited the highest transformation activity toward biphenyl and naphthalene respectively, and also attacked dibenzofuran and dibenzo-p-dioxin. The wide substrate preference of EtbA/EbdA dioxygenase suggested that it plays a more important role in polychlorinated biphenyl (PCB) degradation than does BphA dioxygenase.

A gram-positive strong polychlorinated biphenyl (PCB) degrader, Rhodococcus sp. strain RHA1, can degrade PCBs by cometabolism with biphenyl or ethylbenzene. In RHA1, three sets of aromatic-ring-hydroxylating dioxygenase genes are induced by biphenyl. The large and small subunits of their terminal dioxygenase components are encoded by bphA1 and bphA2, etbA1 and etbA2, and ebdA1 and ebdA2, respectively, and the deduced amino acid sequences of etbA1 and etbA2 are identical to those of ebdA1 and ebdA2, respectively. In this study, we examined the involvement of the respective subunit genes in biphenyl/PCB degradation by RHA1. Reverse transcription-PCR and two-dimensional polyacrylamide gel electrophoresis analyses indicated the induction of RNA and protein products of etbA1 and ebdA1 by biphenyl. Single-and double-disruption mutants of etbA1, ebdA1, and bphA1 were constructed by insertional inactivation. The 4-chlorobiphenyl (4-CB) degradation activities of all the mutants were lower than that of RHA1. The results indicated that all of these genes are involved in biphenyl/PCB degradation. Furthermore, we constructed disruption mutants of ebdA3 and bphA3, encoding ferredoxin, and etbA4, encoding ferredoxin reductase components. The 4-CB degradation activities of these mutants were also lower than that of RHA1, suggesting that all of these genes play a role in biphenyl/PCB degradation. The substrate preferences of etbA1A2/ebdA1A2-and bphA1A2-encoded dioxygenases for PCB congeners were examined using the corresponding mutants. The results indicated that these dioxygenase isozymes have different substrate preferences and that the etbA1A2/ebdA1A2-encoded isozyme is more active on highly chlorinated congeners than the bphA1A2-encoded one.Polychlorinated biphenyls (PCBs) are synthetic compounds that have excellent chemical stability, insulation properties, and resistance to combustion, and they have been used in a wide range of industrial applications. However, since the revelation that PCBs have strong toxicity and are recalcitrant in the environment, environmental contamination by PCBs has become a serious worldwide problem. Degradation of PCBs by microorganisms has been regarded as an effective tool for removal of PCBs from the environment, and many kinds of bacteria which degrade PCBs aerobically have been isolated and characterized (2,4,7,11,14). These bacteria degrade PCBs by cometabolism, with PCBs being degraded via the biphenyl catabolic pathway in the presence of biphenyl (5, 10). In the initial step of the biphenyl catabolic pathway, biphenyl is transformed to 2,3-dihydroxy-1-phenylcyclohexa-4,6-diene (dihydrodiol compound) by an aromatic-ring-hydroxylating dioxygenase (RHDO), biphenyl 2,3-dioxygenase (BDO) (Fig. 1A), which consists of the large and small subunits of terminal oxygenase, ferredoxin, and ferredoxin reductase. BDO is a key enzyme that primarily determines the substrate specificity of PCB degradation.Rhodococcus sp. strain RHA1 is a gram-positive strong PCB degrader. RHA1 can degrade mono-to octachl...

p-Hydroxybenzoate hydroxylase (PHBH) is a flavoprotein monooxygenase that catalyzes the hydroxylation of p-hydroxybenzoate (p-OHB) to 3,4-dihydroxybenzoate (3,4-DOHB). PHBH can bind to other benzoate derivatives in addition to p-OHB; however, hydroxylation does not occur on 3,4-DOHB. Replacement of Tyr385 with Phe forms a mutant, which enables the production of 3,4,5-trihydroxybenzonate (gallic acid) from 3,4-DOHB, although the catalytic activity of the mutant is quite low. In this study, we report how the L199V/Y385F double mutant exhibits activity for producing gallic acid 4.3-fold higher than that of the Y385F single mutant. This improvement in catalytic activity is primarily due to the suppression of a shunt reaction that wastes reduced nicotinamide adenine dinucleotide phosphate by producing H2O2. To further elucidate the molecular mechanism underlying this higher catalytic activity, we performed molecular dynamics simulations and quantum mechanics/molecular mechanics calculations, in addition to determining the crystal structure of the Y385F·3,4-DOHB complex. The simulations showed that the Y385F mutation facilitates the deprotonation of the 4-hydroxy group of 3,4-DOHB, which is necessary for initiating hydroxylation. Moreover, the L199V mutation in addition to the Y385F mutation allows the OH moiety in the peroxide group of C-(4a)-flavin hydroperoxide to come into the proximity of the C5 atom of 3,4-DOHB. Overall, this study provides a consistent explanation for the change in the catalytic activity of PHBH caused by mutations, which will enable us to better design an enzyme with different activities.

The Laser Interferometer Gravitational-wave Observatory Scientific Collaboration and Virgo Collaboration (LVC) sent out 56 gravitational-wave (GW) notices during the third observing run (O3). Japanese collaboration for Gravitational wave ElectroMagnetic follow-up (J-GEM) performed optical and near-infrared observations to identify and observe an electromagnetic (EM) counterpart. We constructed web-based system which enabled us to obtain and share information of candidate host galaxies for the counterpart, and status of our observations. Candidate host galaxies were selected from the GLADE catalog with a weight based on the three-dimensional GW localization map provided by LVC. We conducted galaxy-targeted and wide-field blind surveys, real-time data analysis, and visual inspection of observed galaxies. We performed galaxy-targeted follow-ups to 23 GW events during O3, and the maximum probability covered by our observations reached to 9.8%. Among them, we successfully started observations for 10 GW events within 0.5 days after the detection. This result demonstrates that our followup observation has a potential to constrain EM radiation models for a merger of binary neutron stars at a distance of up to ∼100 Mpc with a probability area of ≤ 500 deg2.

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