Hyperthermophilic flavin reductase from Sulfolobus solfataricus P2: Production and biochemical characterization

2019 ◽  
Vol 66 (6) ◽  
pp. 915-923
Author(s):  
Gokhan Gun ◽  
Rahmi Imamoglu ◽  
Ozge Tatli ◽  
Yuda Yurum ◽  
Ahmet Tarik Baykal ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Sulfolobus Solfataricus ◽  
Flavin Reductase

scholarly journals A novel DNA helicase with strand-annealing activity from the crenarchaeon Sulfolobus solfataricus

2007 ◽  
Vol 408 (1) ◽  
pp. 87-95 ◽  
Author(s):  
Mariarita De Felice ◽  
Valentina Aria ◽  
Luca Esposito ◽  
Mariarosaria De Falco ◽  
Biagio Pucci ◽  
...  
Keyword(s):  
High Efficiency ◽  
Biochemical Characterization ◽  
Sequence Similarity ◽  
Sulfolobus Solfataricus ◽  
Genetic Material ◽  
Dna Helicase ◽  
Soluble Form ◽  
Monomeric Form ◽  
Genome Database ◽  
Strand Annealing

To protect their genetic material cells adopt different mechanisms linked to DNA replication, recombination and repair. Several proteins function at the interface of these DNA transactions. In the present study, we report on the identification of a novel archaeal DNA helicase. BlastP searches of the Sulfolobus solfataricus genome database allowed us to identify an open reading frame (SSO0112, 875 amino acid residues) having sequence similarity with the human RecQ5β. The corresponding protein, termed Hel112 by us, was produced in Escherichia coli in soluble form, purified to homogeneity and characterized. Gel-filtration chromatography and glycerol-gradient sedimentation analyses revealed that Hel112 forms monomers and dimers in solution. Biochemical characterization of the two oligomeric species revealed that only the monomeric form has an ATP-dependent 3′–5′ DNA-helicase activity, whereas, unexpectedly, both the monomeric and dimeric forms possess DNA strand-annealing capability. The Hel112 monomeric form is able to unwind forked and 3′-tailed DNA structures with high efficiency, whereas it is almost inactive on blunt-ended duplexes and bubble-containing molecules. This analysis reveals that S. solfataricus Hel112 shares some enzymatic features with the RecQ-like DNA helicases and suggests potential cellular functions of this protein.

scholarly journals Pyrimidine-specific ribonucleoside hydrolase from the archaeon Sulfolobus solfataricus- biochemical characterization and homology modeling

FEBS Journal ◽  
2008 ◽  
Vol 275 (8) ◽  
pp. 1900-1914 ◽  
Author(s):  
Marina Porcelli ◽  
Luigi Concilio ◽  
Iolanda Peluso ◽  
Anna Marabotti ◽  
Angelo Facchiano ◽  
...  
Keyword(s):  
Homology Modeling ◽  
Biochemical Characterization ◽  
Sulfolobus Solfataricus

Biochemical characterization and mutational improvement of a thermophilic esterase from Sulfolobus solfataricus P2

2010 ◽  
Vol 32 (8) ◽  
pp. 1151-1157 ◽  
Author(s):  
Yu-Shuan Shang ◽  
Xian-En Zhang ◽  
Xu-De Wang ◽  
Yong-Chao Guo ◽  
Zhi-Ping Zhang ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Sulfolobus Solfataricus ◽  
Thermophilic Esterase

scholarly journals Structural and biochemical characterization of EDTA monooxygenase and its physical interaction with a partner flavin reductase

2016 ◽  
Vol 100 (6) ◽  
pp. 989-1003 ◽  
Author(s):  
Se-Young Jun ◽  
Kevin M. Lewis ◽  
Buhyun Youn ◽  
Luying Xun ◽  
ChulHee Kang
Keyword(s):  
Biochemical Characterization ◽  
Physical Interaction ◽  
Flavin Reductase

Biochemical characterization of biliverdins IXβ/δ generated by a selective heme oxygenase

2020 ◽  
Vol 477 (3) ◽  
pp. 601-614
Author(s):  
Beibei Zhang ◽  
Natasha M. Nesbitt ◽  
Pedro José Barbosa Pereira ◽  
Wadie F. Bahou
Keyword(s):  
Biochemical Characterization ◽  
Protoporphyrin Ix ◽  
Kinetic Studies ◽  
Binding Pocket ◽  
Structural Features ◽  
Flavin Reductase ◽  
Reductase Inhibitors ◽  
Free Heme ◽  
Potential Applicability ◽  
Regulated Functions

The pro-oxidant effect of free heme (Fe2+-protoporphyrin IX) is neutralized by phylogenetically-conserved heme oxygenases (HMOX) that generate carbon monoxide, free ferrous iron, and biliverdin (BV) tetrapyrrole(s), with downstream BV reduction by non-redundant NADPH-dependent BV reductases (BLVRA and BLVRB) that retain isomer-restricted functional activity for bilirubin (BR) generation. Regioselectivity for the heme α-meso carbon resulting in predominant BV IXα generation is a defining characteristic of canonical HMOXs, thereby limiting generation and availability of BVs IXβ, IXδ, and IXγ as BLVRB substrates. We have now exploited the unique capacity of the Pseudomonas aeruginosa (P. aeruginosa) hemO/pigA gene for focused generation of isomeric BVs (IXβ and IXδ). A scalable system followed by isomeric separation yielded highly pure samples with predicted hydrogen-bonded structure(s) as documented by 1H NMR spectroscopy. Detailed kinetic studies established near-identical activity of BV IXβ and BV IXδ as BLVRB-selective substrates, with confirmation of an ordered sequential mechanism of BR/NADP+ dissociation. Halogenated xanthene-based compounds previously identified as BLVRB-targeted flavin reductase inhibitors displayed comparable inhibition parameters using BV IXβ as substrate, documenting common structural features of the cofactor/substrate-binding pocket. These data provide further insights into structure/activity mechanisms of isomeric BVs as BLVRB substrates, with potential applicability to further dissect redox-regulated functions in cytoprotection and hematopoiesis.

scholarly journals Purification and biochemical characterization of a poly(ADP-ribose) polymerase-like enzyme from the thermophilic archaeon Sulfolobus solfataricus

1998 ◽  
Vol 335 (2) ◽  
pp. 441-447 ◽  
Author(s):  
Maria Rosaria FARAONE-MENNELLA ◽  
Agata GAMBACORTA ◽  
Barbara NICOLAUS ◽  
Benedetta FARINA
Keyword(s):  
Biochemical Characterization ◽  
Sulfolobus Solfataricus ◽  
Enzymic Activity ◽  
Sds Page ◽  
Thermophilic Archaeon ◽  
Displacement Assay ◽  
Elongation Step ◽  
Ph Range ◽  
Good Agreement

A poly(ADP-ribose) polymerase-like enzyme, detected in a crude homogenate from Sulfolobus solfataricus by means of activity and immunoblot analyses, was purified to electrophoretic homogeneity by a rapid procedure including two sequential affinity chromatographies, on NAD+-agarose and DNA-Sepharose. The latter column selected specifically the poly(ADP-ribosyl)ating enzyme with a 17% recovery of enzymic activity and a purification of more than 15000-fold. The molecular mass (54–55 kDa) assessed by SDS/PAGE and immunoblot was definitely lower than that determined for the corresponding eukaryotic protein. The enzyme was proved to be thermophilic, with a temperature optimum of approx. 80 °C, and thermostable, with a half-life of 204 min at 80 °C, in good agreement with the requirements of a thermozyme. It displayed a Km towards NAD+ of 154±50 µM; in the pH range 6.5–10.0 the activity values were similar, not showing a real optimum pH. The enzyme was able to bind homologous DNA, as evidenced by the ethidium bromide displacement assay. The product of the ADP-ribosylating reaction co-migrated with the short oligomers of ADP-ribose (less than 6 residues) from a eukaryotic source. Reverse-phase HPLC analysis of the products, after digestion with phosphodiesterase I, gave an elution profile reproducing that obtained by the enzymic digestion of the rat testis poly(ADP-ribose). These results strongly suggest that the activities of the purified enzyme include the elongation step.

scholarly journals Genetic and Biochemical Characterization of a 2,4,6-Trichlorophenol Degradation Pathway in Ralstonia eutropha JMP134

2002 ◽  
Vol 184 (13) ◽  
pp. 3492-3500 ◽  
Author(s):  
Tai Man Louie ◽  
Christopher M. Webster ◽  
Luying Xun
Keyword(s):  
Sequence Analysis ◽  
Biochemical Characterization ◽  
Ralstonia Eutropha ◽  
Degradation Pathway ◽  
Partial Purification ◽  
Flavin Reductase ◽  
Catabolic Repression ◽  
Cell Extracts ◽  
Insertional Inactivation

ABSTRACT Ralstonia eutropha JMP134 can grow on several chlorinated aromatic pollutants, including 2,4-dichlorophenoxyacetate and 2,4,6-trichlorophenol (2,4,6-TCP). Although a 2,4,6-TCP degradation pathway in JMP134 has been proposed, the enzymes and genes responsible for 2,4,6-TCP degradation have not been characterized. In this study, we found that 2,4,6-TCP degradation by JMP134 was inducible by 2,4,6-TCP and subject to catabolic repression by glutamate. We detected 2,4,6-TCP-degrading activities in JMP134 cell extracts. Our partial purification and initial characterization of the enzyme indicated that a reduced flavin adenine dinucleotide (FADH2)-utilizing monooxygenase converted 2,4,6-TCP to 6-chlorohydroxyquinol (6-CHQ). The finding directed us to PCR amplify a 3.2-kb fragment containing a gene cluster (tcpABC) from JMP134 by using primers designed from conserved regions of FADH2-utilizing monooxygenases and hydroxyquinol 1,2-dioxygenases. Sequence analysis indicated that tcpA, tcpB, and tcpC encoded an FADH2-utilizing monooxygenase, a probable flavin reductase, and a 6-CHQ 1,2-dioxygenase, respectively. The three genes were individually inactivated in JMP134. The tcpA mutant failed to degrade 2,4,6-TCP, while both tcpB and tcpC mutants degraded 2,4,6-TCP to an oxidized product of 6-CHQ. Insertional inactivation of tcpB may have led to a polar effect on downstream tcpC, and this probably resulted in the accumulation of the oxidized form of 6-CHQ. For further characterization, TcpA was produced, purified, and shown to transform 2,4,6-TCP to 6-CHQ when FADH2 was supplied by an Escherichia coli flavin reductase. TcpC produced in E. coli oxidized 6-CHQ to 2-chloromaleylacetate. Thus, our data suggest that JMP134 transforms 2,4,6-TCP to 2-chloromaleylacetate by TcpA and TcpC. Sequence analysis suggests that tcpB may function as an FAD reductase, but experimental data did not support this hypothesis. The function of TcpB remains unknown.

Biochemical characterization of two Cdc6/ORC1-like proteins from the crenarchaeon Sulfolobus solfataricus

Extremophiles ◽  
2005 ◽  
Vol 10 (1) ◽  
pp. 61-70 ◽  
Author(s):  
Mariarita De Felice ◽  
Luca Esposito ◽  
Mosè Rossi ◽  
Francesca M. Pisani
Keyword(s):  
Biochemical Characterization ◽  
Sulfolobus Solfataricus
Sign in / Sign up

Export Citation Format

Share Document