Изменения четвертичной структуры нуклеотид-регулируемой пирофосфатазы из Desulfitobacterium hafniense при взаимодействии с лигандами, выявленные методом малоуглового рентгеновского рассеяния в растворе

2020 ◽  
Vol 65 (5) ◽  
pp. 780-785
Author(s):  
Л. А. Дадинова ◽  
В. А. Анашкин ◽  
Э. В. Штыкова
2015 ◽  
Vol 10 (1) ◽  
Author(s):  
Tobias Goris ◽  
Bastian Hornung ◽  
Thomas Kruse ◽  
Anika Reinhold ◽  
Martin Westermann ◽  
...  

2012 ◽  
Vol 78 (22) ◽  
pp. 8025-8032 ◽  
Author(s):  
Anika Reinhold ◽  
Martin Westermann ◽  
Jana Seifert ◽  
Martin von Bergen ◽  
Torsten Schubert ◽  
...  

ABSTRACTCorrinoids are essential cofactors of reductive dehalogenases in anaerobic bacteria. Microorganisms mediating reductive dechlorination as part of their energy metabolism are either capable ofde novocorrinoid biosynthesis (e.g.,Desulfitobacteriumspp.) or dependent on exogenous vitamin B12(e.g.,Dehalococcoidesspp.). In this study, the impact of exogenous vitamin B12(cyanocobalamin) and of tetrachloroethene (PCE) on the synthesis and the subcellular localization of the reductive PCE dehalogenase was investigated in the Gram-positiveDesulfitobacterium hafniensestrain Y51, a bacterium able to synthesize corrinoidsde novo. PCE-depleted cells grown for several subcultivation steps on fumarate as an alternative electron acceptor lost the tetrachloroethene-reductive dehalogenase (PceA) activity by the transposition of thepcegene cluster. In the absence of vitamin B12, a gradual decrease of the PceA activity and protein amount was observed; after 5 subcultivation steps with 10% inoculum, more than 90% of the enzyme activity and of the PceA protein was lost. In the presence of vitamin B12, a significant delay in the decrease of the PceA activity with an ∼90% loss after 20 subcultivation steps was observed. This corresponded to the decrease in thepceAgene level, indicating that exogenous vitamin B12hampered the transposition of thepcegene cluster. In the absence or presence of exogenous vitamin B12, the intracellular corrinoid level decreased in fumarate-grown cells and the PceA precursor formed catalytically inactive, corrinoid-free multiprotein aggregates. The data indicate that exogenous vitamin B12is not incorporated into the PceA precursor, even though it affects the transposition of thepcegene cluster.


2006 ◽  
Vol 188 (7) ◽  
pp. 2604-2613 ◽  
Author(s):  
Krisztina Gábor ◽  
Carla S. Veríssimo ◽  
Barbara C. Cyran ◽  
Paul ter Horst ◽  
Nienke P. Meijer ◽  
...  

ABSTRACT The recently identified CprK branch of the CRP (cyclic AMP receptor protein)-FNR (fumarate and nitrate reduction regulator) family of transcriptional regulators includes proteins that activate the transcription of genes encoding proteins involved in reductive dehalogenation of chlorinated aromatic compounds. Here we report the characterization of the CprK1 protein from Desulfitobacterium hafniense, an anaerobic low-G+C gram-positive bacterium that is capable of reductive dechlorination of 3-chloro-4-hydroxyphenylacetic acid (Cl-OHPA). The gene encoding CprK1 was cloned and functionally overexpressed in Escherichia coli, and the protein was subsequently purified to homogeneity. To investigate the interaction of CprK1 with three of its predicted binding sequences (dehaloboxes), we performed in vitro DNA-binding assays (electrophoretic mobility shift assays) as well as in vivo promoter probe assays. Our results show that CprK1 binds its target dehaloboxes with high affinity (dissociation constant, 90 nM) in the presence of Cl-OHPA and that transcriptional initiation by CprK1 is influenced by deviations in the dehaloboxes from the consensus TTAAT----ATTAA sequence. A mutant CprK1 protein was created by a Val→Glu substitution at a conserved position in the recognition α-helix that gained FNR-type DNA-binding specificity, recognizing the TTGAT----ATCAA sequence (FNR box) instead of the dehaloboxes. CprK1 was subject to oxidative inactivation in vitro, most likely caused by the formation of an intermolecular disulfide bridge between Cys11 and Cys200. The possibility of redox regulation of CprK1 by a thiol-disulfide exchange reaction was investigated by using two Cys→Ser mutants. Our results indicate that a Cys11-Cys200 disulfide bridge does not appear to play a physiological role in the regulation of CprK1.


2006 ◽  
Vol 72 (9) ◽  
pp. 5998-6003 ◽  
Author(s):  
Taiki Futagami ◽  
Takehito Yamaguchi ◽  
Shun-ichi Nakayama ◽  
Masatoshi Goto ◽  
Kensuke Furukawa

ABSTRACT The dehalorespiring Desulfitobacterium hafniense strain Y51 efficiently dechlorinates tetrachloroethene (PCE) to cis-1,2-dichloroethene (cis-DCE) via trichloroethene by PceA reductive dehalogenase encoded by the pceA gene. In a previous study, we found that the significant growth inhibition of strain Y51 occurred in the presence of commercial cis-DCE. In this study, it turned out that the growth inhibition was caused by chloroform (CF) contamination of cis-DCE. Interestingly, CF did not affect the growth of PCE-nondechlorinating SD (small deletion) and LD (large deletion) variants, where the former fails to transcribe the pceABC genes caused by a deletion of the promoter and the latter lost the entire pceABCT gene cluster. Therefore, PCE-nondechlorinating variants, mostly LD variant, became predominant, and dechlorination activity was significantly reduced in the presence of CF. Moreover, such a growth inhibitory effect was also observed in the presence of carbon tetrachloride at 1 μM, but not carbon dichloride even at 1 mM.


2016 ◽  
Vol 473 (14) ◽  
pp. 2097-2107 ◽  
Author(s):  
Viktor A. Anashkin ◽  
Anu Salminen ◽  
Natalia N. Vorobjeva ◽  
Reijo Lahti ◽  
Alexander A. Baykov

Many prokaryotic soluble PPases (pyrophosphatases) contain a pair of regulatory adenine nucleotide-binding CBS (cystathionine β-synthase) domains that act as ‘internal inhibitors’ whose effect is modulated by nucleotide binding. Although such regulatory domains are found in important enzymes and transporters, the underlying regulatory mechanism has only begun to come into focus. We reported previously that CBS domains bind nucleotides co-operatively and induce positive kinetic co-operativity (non-Michaelian behaviour) in CBS-PPases (CBS domain-containing PPases). In the present study, we demonstrate that a homodimeric ehPPase (Ethanoligenens harbinense PPase) containing an inherent mutation in an otherwise conserved asparagine residue in a loop near the active site exhibits non-co-operative hydrolysis kinetics. A similar N312S substitution in ‘co-operative’ dhPPase (Desulfitobacterium hafniense PPase) abolished kinetic co-operativity while causing only minor effects on nucleotide-binding affinity and co-operativity. However, the substitution reversed the effect of diadenosine tetraphosphate, abolishing kinetic co-operativity in wild-type dhPPase, but restoring it in the variant dhPPase. A reverse serine-to-asparagine replacement restored kinetic co-operativity in ehPPase. Molecular dynamics simulations revealed that the asparagine substitution resulted in a change in the hydrogen-bonding pattern around the asparagine residue and the subunit interface, allowing greater flexibility at the subunit interface without a marked effect on the overall structure. These findings identify this asparagine residue as lying at the ‘crossroads’ of information paths connecting catalytic and regulatory domains within a subunit and catalytic sites between subunits.


2012 ◽  
Author(s):  
Kim Sang-Hoon ◽  
Christina Hardzman ◽  
John k. Davis ◽  
Rachel Hutcheson ◽  
Joan B. Broderick ◽  
...  

1999 ◽  
Vol 65 (12) ◽  
pp. 5212-5221 ◽  
Author(s):  
Jan Gerritse ◽  
Oliver Drzyzga ◽  
Geert Kloetstra ◽  
Mischa Keijmel ◽  
Luit P. Wiersum ◽  
...  

ABSTRACT Strain TCE1, a strictly anaerobic bacterium that can grow by reductive dechlorination of tetrachloroethene (PCE) and trichloroethene (TCE), was isolated by selective enrichment from a PCE-dechlorinating chemostat mixed culture. Strain TCE1 is a gram-positive, motile, curved rod-shaped organism that is 2 to 4 by 0.6 to 0.8 μm and has approximately six lateral flagella. The pH and temperature optima for growth are 7.2 and 35°C, respectively. On the basis of a comparative 16S rRNA sequence analysis, this bacterium was identified as a new strain of Desulfitobacterium frappieri, because it exhibited 99.7% relatedness to the D. frappieri type strain, strain PCP-1. Growth with H2, formate,l-lactate, butyrate, crotonate, or ethanol as the electron donor depends on the availability of an external electron acceptor. Pyruvate and serine can also be used fermentatively. Electron donors (except formate and H2) are oxidized to acetate and CO2. When l-lactate is the growth substrate, strain TCE1 can use the following electron acceptors: PCE and TCE (to produce cis-1,2-dichloroethene), sulfite and thiosulfate (to produce sulfide), nitrate (to produce nitrite), and fumarate (to produce succinate). Strain TCE1 is not able to reductively dechlorinate 3-chloro-4-hydroxyphenylacetate. The growth yields of the newly isolated bacterium when PCE is the electron acceptor are similar to those obtained for other dehalorespiring anaerobes (e.g.,Desulfitobacterium sp. strain PCE1 andDesulfitobacterium hafniense) and the maximum specific reductive dechlorination rates are 4 to 16 times higher (up to 1.4 μmol of chloride released · min−1 · mg of protein−1). Dechlorination of PCE and TCE is an inducible process. In PCE-limited chemostat cultures of strain TCE1, dechlorination is strongly inhibited by sulfite but not by other alternative electron acceptors, such as fumarate or nitrate.


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