Structural Characterization of Pandoraea pnomenusa B-356 Biphenyl Dioxygenase Reveals Features of Potent Polychlorinated Biphenyl-Degrading Enzymes

The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase $(BPDO_{B356})$. $BPDO_{B356}$, a heterohexameric $(αβ)_3$ Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of $BPDO_{B356}$ with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of $BPDO_{B356}$ and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2′-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments

Kilonova Luminosity Function Constraints Based on Zwicky Transient Facility Searches for 13 Neutron Star Merger Triggers during O3

We present a systematic search for optical counterparts to 13 gravitational wave (GW) triggers involving at least one neutron star during LIGO/Virgo's third observing run (O3). We searched binary neutron star (BNS) and neutron star black hole (NSBH) merger localizations with the Zwicky Transient Facility (ZTF) and undertook follow-up with the Global Relay of Observatories Watching Transients Happen (GROWTH) collaboration. The GW triggers had a median localization area of 4480 deg², a median distance of 267 Mpc, and false-alarm rates ranging from 1.5 to 10⁻²⁵ yr⁻¹. The ZTF coverage in the g and r bands had a median enclosed probability of 39%, median depth of 20.8 mag, and median time lag between merger and the start of observations of 1.5 hr. The O3 follow-up by the GROWTH team comprised 340 UltraViolet/Optical/InfraRed (UVOIR) photometric points, 64 OIR spectra, and three radio images using 17 different telescopes. We find no promising kilonovae (radioactivity-powered counterparts), and we show how to convert the upper limits to constrain the underlying kilonova luminosity function. Initially, we assume that all GW triggers are bona fide astrophysical events regardless of false-alarm rate and that kilonovae accompanying BNS and NSBH mergers are drawn from a common population; later, we relax these assumptions. Assuming that all kilonovae are at least as luminous as the discovery magnitude of GW170817 (−16.1 mag), we calculate that our joint probability of detecting zero kilonovae is only 4.2%. If we assume that all kilonovae are brighter than −16.6 mag (the extrapolated peak magnitude of GW170817) and fade at a rate of 1 mag day⁻¹ (similar to GW170817), the joint probability of zero detections is 7%. If we separate the NSBH and BNS populations based on the online classifications, the joint probability of zero detections, assuming all kilonovae are brighter than −16.6 mag, is 9.7% for NSBH and 7.9% for BNS mergers. Moreover, no more than 10⁻⁴, or φ > 30° to be consistent with our limits. We look forward to searches in the fourth GW observing run; even 17 neutron star mergers with only 50% coverage to a depth of −16 mag would constrain the maximum fraction of bright kilonovae to <25%

Identification of the Dimer Interface of a Bacterial Ca²⁺/H⁺ Antiporter

Members of the calcium/cation antiporter superfamily, including the cardiac sodium/calcium exchangers, are secondary active transporters that play an essential role in cellular Ca²⁺ homeostasis. A notable feature of this group of transporters is the high levels of sequence similarity in relatively short sequences constituting the functionally important α-1 and α-2 regions in contrast to relatively lower degrees of similarity in the extended adjoining sequences. This suggests a similar structure and function of core transport machinery but possible differences in topology and/or oligomerization, a topic that has not been adequately addressed. Here we present the first example of purification of a bacterial member of this superfamily (CAX^CK31) and analyze its quaternary structure. Purification of CAX^CK31 required the presence of a choline headgroup-containing detergent or lipid to yield stable preparations of the monomeric transporter. H⁺-driven Ca²⁺ transport was demonstrated by reconstituting purified CAX^CK31 into liposomes. Dimeric CAX^CK31 could be isolated by manipulation of detergent micelles. Dimer formation was shown to be dependent on micelle composition as well as protein concentration. Furthermore, we establish that CAX^CK31 forms dimers in the membrane by analysis of cross-linked proteins. Using a dimeric homology model derived from the monomeric structure of the archaeal NCX homologue (Protein Data Bank entry 3V5U), we introduced cysteine residues and through cross-linking experiments established the role of transmembrane helices 2 and 6 in the putative dimer interface

Anaerobic crystallization and initial X-ray diffraction data of biphenyl 2,3-dioxygenase from Burkholderia xenovorans LB400: addition of agarose improved the quality of the crystals

Biphenyl 2,3-dioxygenase from B. xenovorans LB400 and its variants BPDOP4 and BPDORR41 were crystallized using agarose gel and the crystals were characterized using X-ray diffraction

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

Biphenyl dioxygenase (BPDO) catalyzes the aerobic transformation of biphenyl and various polychlorinated biphenyls (PCBs). In three different assays, BPDOB356 from Pandoraea pnomenusa B-356 was a more potent PCB-degrading enzyme than BPDOLB400 from Burkholderia xenovorans LB400 (75% amino acid sequence identity), transforming nine congeners in the following order of preference: 2,3′,4-trichloro ∼ 2,3,4′-trichloro > 3,3′-dichloro > 2,4,4′-trichloro > 4,4′-dichloro ∼ 2,2′-dichloro > 2,6-dichloro > 2,2′,3,3′-tetrachloro ∼ 2,2′,5,5′-tetrachloro. Except for 2,2′,5,5′-tetrachlorobiphenyl, BPDOB356 transformed each congener at a higher rate than BPDOLB400. The assays used either whole cells or purified enzymes and either individual congeners or mixtures of congeners. Product analyses established previously unrecognized BPDOB356 activities, including the 3,4-dihydroxylation of 2,6-dichlorobiphenyl. BPDOLB400 had a greater apparent specificity for biphenyl than BPDOB356 (kcat/Km = 2.4 × 106 ± 0.7 × 106 M−1 s−1 versus kcat/Km = 0.21 × 106 ± 0.04 × 106 M−1 s−1). However, the latter transformed biphenyl at a higher maximal rate (kcat = 4.1 ± 0.2 s−1 versus kcat = 0.4 ± 0.1 s−1). A variant of BPDOLB400 containing four active site residues of BPDOB356 transformed para-substituted congeners better than BPDOLB400. Interestingly, a substitution remote from the active site, A267S, increased the enzyme's preference for meta-substituted congeners. Moreover, this substitution had a greater effect on the kinetics of biphenyl utilization than substitutions in the substrate-binding pocket. In all variants, the degree of coupling between congener depletion and O2 consumption was approximately proportional to congener depletion. At 2.4-Å resolution, the crystal structure of the BPDOB356-2,6-dichlorobiphenyl complex, the first crystal structure of a BPDO-PCB complex, provided additional insight into the reactivity of this isozyme with this congener, as well as into the differences in congener preferences of the BPDOs