Detection of C-P-lyase activity in a cell-free extract of Escherichia coli

Кононова, С. В.; Trutko, S. M.; Лауринавичус, К. С.

doi:10.1134/s0003683807040060

Cited by 6 publications

(3 citation statements)

References 20 publications

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“…5, reaction ii). Formation of methane from methylphosphonate has been previously demonstrated in vitro and was found to be stimulated by ATP and NADH, although no intermediates were identified (25).…”

Section: Discussionmentioning

confidence: 93%

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

Jochimsen

Lolle

McSorley

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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Organophosphonate utilization by Escherichia coli requires the 14 cistrons of the phnCDEFGHIJKLMNOP operon, of which the carbon-phosphorus lyase has been postulated to consist of the seven polypeptides specified by phnG to phnM . A 5,660-bp DNA fragment encompassing phnGHIJKLM is cloned, followed by expression in E. coli and purification of Phn-polypeptides. PhnG, PhnH, PhnI, PhnJ, and PhnK copurify as a protein complex by ion-exchange, size-exclusion, and affinity chromatography. The five polypeptides also comigrate in native-PAGE. Cross-linking of the purified protein complex reveals a close proximity of PhnG, PhnI, PhnJ, and PhnK, as these subunits disappear concomitant with the formation of large cross-linked protein complexes. Two molecular forms are identified, a major form of molecular mass of approximately 260 kDa, a minor form of approximately 640 kDa. The stoichiometry of the protein complex is suggested to be PhnG 4 H 2 I 2 J 2 K. Deletion of individual phn genes reveals that a strain harboring plasmid-borne phnGHIJ produces a protein complex consisting of PhnG, PhnH, PhnI, and PhnJ, whereas a strain harboring plasmid-borne phnGIJK produces a protein complex consisting of PhnG and PhnI. We conclude that phnGHIJK specify a soluble multisubunit protein complex essential for organophosphonate utilization.

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Section: Discussionmentioning

confidence: 93%

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

Jochimsen

Lolle

McSorley

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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“…In this enterobacterium, alkylphosphonic acids initially react with an ATP molecule; the product is then dephosphorylated, with the release of pyrophosphate and an intermediate that in turn undergoes cyclization, yielding a 5′‐phospho‐α‐D‐ribosyl 1,2‐cyclic phosphonate. The latter is further metabolized by other hydrolases, leading to the final release of diphosphate and phosphate ions (Mazumder, ; Kononova et al ., ; Kamat et al ., ; Hove‐Jensen et al ., ). Although the presence of ribosyl intermediates in A. variabilis extracts was not definitely demonstrated due to their presumably low concentrations and the unavailability of pure standards, the peak at 15.4 ppm may be most likely attributed to 5′‐phospho‐α‐D‐ribosyl 1′‐( N ‐acetylaminomethylephosphonic acid).…”

Section: Resultsmentioning

confidence: 98%

Biodegradation of the aminopolyphosphonate DTPMP by the cyanobacterium Anabaena variabilis proceeds via a C–P lyase‐independent pathway

Drzyzga

Forlani

Vermander

et al. 2017

Environmental Microbiology

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Cyanobacteria, the only prokaryotes capable of oxygenic photosynthesis, play a major role in carbon, nitrogen and phosphorus global cycling. Under conditions of increased P availability and nutrient loading, some cyanobacteria are capable of blooming, rapidly multiplying and possibly altering the ecological structure of the ecosystem. Because of their ability of using non-conventional P sources, these microalgae can be used for bioremediation purposes. Under this perspective, the metabolization of the polyphosphonate diethylenetriaminepenta(methylenephosphonic) acid (DTPMP) by the strain CCALA 007 of Anabaena variabilis was investigated using P NMR analysis. Results showed a quantitative breakdown of DTPMP by cell-free extracts from cyanobacterial cells grown in the absence of any phosphonate. The identification of intermediates and products allowed us to propose a unique and new biodegradation pathway in which the formation of (N-acetylaminomethyl)phosphonic acid represents a key step. This hypothesis was strengthened by the results obtained by incubating cell-free extracts with pathway intermediates. When Anabaena cultures were grown in the presence of the phosphonate, or phosphorus-starved before the extraction, significantly higher biodegradation rates were found.

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“…Mutational studies have suggested that PhnP is required in the presence of a functional phnN product for phosphonate utilization (Metcalf & Wanner, 1993). Attempts to detect C-P lyase activity in a cell-free system have been inconsistent, possibly owing to the fact that the enzyme complex is predicted to be membrane-associated (Kononova et al, 2007;Ternan et al, 1998). Consequently, the reaction mechanism of enzymatic C-P bond cleavage is poorly understood.…”

Section: Introductionmentioning

confidence: 99%

Expression, purification and preliminary diffraction studies of PhnP

Podzelinska

Soares

et al. 2008

Acta Cryst Sect F

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PhnP belongs to a 14-gene operon that supports the growth of Escherichia coli on alkylphosphonates as a sole source of phosphorus; however, the exact biochemistry of phosphonate degradation by this pathway is poorly understood. The protein was recombinantly expressed in Escherichia coli and purified to homogeneity. Sitting-drop vapour diffusion in combination with microseeding was used to obtain crystals that were suitable for X-ray diffraction. Data were collected to 1.3 Å and the crystals belonged to space group C2, with unit-cell parameters a = 111.65, b = 75.41, c = 83.23 Å , = = 90, = 126.3 .

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Detection of C-P-lyase activity in a cell-free extract of Escherichia coli

Cited by 6 publications

References 20 publications

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

Five phosphonate operon gene products as components of a multi-subunit complex of the carbon-phosphorus lyase pathway

Biodegradation of the aminopolyphosphonate DTPMP by the cyanobacterium Anabaena variabilis proceeds via a C–P lyase‐independent pathway

Expression, purification and preliminary diffraction studies of PhnP

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