Genome Sequence of Nitratireductor pacificus Type Strain pht-3B

Lai, Qiliang; Li, Guizhen; Shao, Zongze

doi:10.1128/jb.01905-12

Cited by 11 publications

(10 citation statements)

References 5 publications

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“…A BLASTp search of the Nitratireductor pacificus pht-3B genome 23 using the protein sequence of Comamonas sp. 7D-2 BhbA (YP_007878394) revealed three putative reductive dehalogenase homologs sharing 72% (WP_008593084.1), 51% (WP_008595008.1) and 42% (WP_008597722.1) identity with BhbA.…”

Section: Methodsmentioning

confidence: 99%

“…The PCR product was cloned into the Bsr GI and Bam HI sites of pP T7 plasmid24 using Infusion HD (Clontech) and transformed into E. coli NEB5α. Once the sequence of the insert was confirmed the purified plasmid was transformed into B. megaterium MS941 containing the pT7-RNAP plasmid that permits xylose inducible expression of T7 polymerase23, using the modified minimal medium protoplast transformation protocol25.…”

Section: Methodsmentioning

confidence: 99%

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Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation

Payne

Quezada

Fisher

et al. 2014

Nature

280

392

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Organohalide chemistry underpins many industrial and agricultural processes, and a large proportion of environmental pollutants are organohalides1. Nevertheless, organohalide chemistry is not exclusively of anthropogenic origin, with natural abiotic and biological processes contributing to the global halide cycle2–3. Reductive dehalogenases are responsible for biological dehalogenation in organohalide respiring bacteria4–5, with substrates including the notorious polychlorinated biphenyls (PCBs) or dioxins6–7. These proteins form a distinct subfamily of cobalamin (B12) dependent enzymes that are usually membrane-associated and oxygen-sensitive, hindering detailed studies8–12. We report the characterisation of a soluble, oxygen-tolerant reductive dehalogenase and, by combining structure determination with EPR spectroscopy and simulation, show that a direct interaction between the cobalamin cobalt and the substrate halogen underpins catalysis. In contrast to the carbon-Co bond chemistry catalyzed by the other cobalamin-dependent subfamilies13 we propose that reductive dehalogenases achieve reduction of the organohalide substrate via halogen-Co bond formation. This presents a new paradigm in both organohalide and cobalamin (bio)chemistry that will guide future exploitation of these enzymes in bioremediation or biocatalysis.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Reductive dehalogenase structure suggests a mechanism for B12-dependent dehalogenation

Payne

Quezada

Fisher

et al. 2014

Nature

280

392

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“…The N. pacificus pht-3b genome contains the N. pacificus reductive dehalogenase WP_008597722.1 gene (NpRdhA), ferredoxin NADPH oxidoreductase WP_008597723.1 gene (Fnr), [2Fe–2S] ferredoxin WP_008597724.1 gene (Fd2), and [4Fe-4S] ferredoxin WP_008597725.1 gene (Fd4). The Nitratireductor sp.…”

Section: Materials and Methodsmentioning

confidence: 99%

“…However, detailed kinetic studies were hampered by the availability of spinach Fnr (SpFnr). The NprdhA gene is situated in a putative operon in the N. pacificus genome that includes a putative ferredoxin-NADPH oxidoreductase and [4Fe–4S] and [2Fe–2S] ferredoxins . This putative redox system associated with the NpRdhA nonrespiratory dehalogenase defines a clear path by which NAD(P)H derived reducing equivalents can be provided to NpRdhA, in contrast to the as yet ill-defined redox partners of the respiratory dehalogenases .…”

mentioning

confidence: 99%

NADPH-Driven Organohalide Reduction by a Nonrespiratory Reductive Dehalogenase

et al. 2018

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Reductive dehalogenases are corrinoid and iron-sulfur cluster-dependent enzymes that mostly act as the terminal oxidoreductases in the bacterial organohalide respiration process. This process often leads to detoxification of recalcitrant organohalide pollutants. While low cell yields and oxygen sensitivity hamper the study of many reductive dehalogenases, this is not the case for the nonrespiratory reductive dehalogenase NpRdhA from Nitratireductor pacificus. We here report in vitro and in vivo reconstitution of an NADPH-dependent reducing system for NpRdhA. Surprisingly, NpRdhA mediated organohalide reduction could not be supported using N. pacificus ferredoxin-NAD(P)H oxidoreductase and associated ferredoxins. Instead, we found a nonphysiological system comprised of the Escherichia coli flavodoxin reductase (EcFldr) in combination with spinach ferredoxin (SpFd) was able to support NADPH-dependent organohalide reduction by NpRdhA. Using this system, organohalide reduction can be performed under both anaerobic and aerobic conditions, with 1.1 ± 0.1 and 3.5 ± 0.3 equiv of NADPH consumed per product produced, respectively. No significant enzyme inactivation under aerobic conditions was observed, suggesting a Co(I) species is unlikely to be present under steady state conditions. Furthermore, reduction of the Co(II) resting state was not observed in the absence of substrate. Only the coexpression of EcFldr, SpFd, and NpRdhA in Bacillus megaterium conferred the latter with the ability to reduce brominated NpRdhA substrates in vivo, in agreement with our in vitro observations. Our work provides new insights into biological reductive dehalogenase reduction and establishes a blueprint for the minimal functional organohalide reduction module required for bioremediation in situ.

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“…The next achievement came with a cometabolic putative reductive dehalogenase, NpRdhA, from Nitratireductor pacificus strain pht-3B, which was heterologously expressed in Bacillus megaterium, also capable of de novo cobalamin biosynthesis [50]. Strain pht-3B was previously isolated from a pyrene-degrading consortium and C-terminal His-tagged NpRdhA without TAT signal and rdhB gene was obtained in an active form in aerobically grown B. megaterium by inducing its well-known xylose-inducible promoter [70]. The recombinant NpRdhA represents a phylogenetically distinct and aerobic RdhA, and it allowed further studies to reveal crystal structure discussed earlier [50].…”

Section: Environmental Applications Of Enzymesmentioning

confidence: 99%