scholarly journals Molecular and biochemical characterization of the thermoactive family 1 pectate lyase from the hyperthermophilic bacterium Thermotoga maritima

2003 ◽  
Vol 370 (2) ◽  
pp. 651-659 ◽  
Author(s):  
Leon D. KLUSKENS ◽  
Gert-Jan W.M. van ALEBEEK ◽  
Alphons G.J. VORAGEN ◽  
Willem M. de VOS ◽  
John van der OOST
Keyword(s):  
Thermal Inactivation ◽  
Biochemical Characterization ◽  
Thermotoga Maritima ◽  
Pectate Lyase ◽  
Major Product ◽  
Degree Of Polymerization ◽  
Extracellular Medium ◽  
Native Form ◽  
Hyperthermophilic Bacterium

The ability of the hyperthermophilic bacterium Thermotoga maritima to grow on pectin as a sole carbon source coincides with the secretion of a pectate lyase A (PelA) in the extracellular medium. The pelA gene of T. maritima was functionally expressed in Escherichia coli as the first heterologously produced thermophilic pectinase, and purified to homogeneity. Gel filtration indicated that the native form of PelA is tetrameric. Highest activity (422units/mg, with a Km of 0.06mM) was demonstrated on polygalacturonic acid (PGA), whereas pectins with an increasing degree of methylation were degraded at a decreasing rate. In the tradition of pectate lyases, PelA demonstrated full dependency on Ca2+ for stability and activity. The enzyme is highly thermoactive and thermostable, operating optimally at 90°C and pH9.0, with a half-life for thermal inactivation of almost 2h at 95°C, and an apparent melting temperature of 102.5°C. Detailed characterization of the product formation with PGA indicated that PelA has a unique eliminative exo-cleavage pattern liberating unsaturated trigalacturonate as the major product, in contrast with unsaturated digalacturonate for other exopectate lyases known. The unique exo-acting mode of action was supported by progression profiles of PelA on oligogalacturonides (degree of polymerization, 3—8) and the examination of the bond cleavage frequencies.

Expression and Characterization of a Novel Nitrilase from Hyperthermophilic Bacterium Thermotoga maritima MSB8

2015 ◽  
Vol 25 (10) ◽  
pp. 1660-1669 ◽  
Author(s):  
Zhi Chen ◽  
Huayou Chen ◽  
Zhong Ni ◽  
Rui Tian ◽  
Tianxi Zhang ◽  
...  
Keyword(s):  
Thermotoga Maritima ◽  
Hyperthermophilic Bacterium

scholarly journals Characterization of an Exceedingly Active NADH Oxidase from the Anaerobic Hyperthermophilic Bacterium Thermotoga maritima

2007 ◽  
Vol 189 (8) ◽  
pp. 3312-3317 ◽  
Author(s):  
Xianqin Yang ◽  
Kesen Ma
Keyword(s):  
Nadh Oxidase ◽  
Specific Activity ◽  
Ph Value ◽  
Thermotoga Maritima ◽  
Cell Extract ◽  
Hyperthermophilic Bacterium ◽  
Its Gene ◽  
Molecular Masses ◽  
New Type

ABSTRACT An NADH oxidase from the anaerobic hyperthermophilic bacterium Thermotoga maritima was purified. The enzyme was very active in catalyzing the reduction of oxygen to hydrogen peroxide with an optimal pH value of 7 at 80°C. The Vmax was 230 ± 14 μmol/min/mg (k cat/Km = 548,000 min−1 mM−1), and the Km values for NADH and oxygen were 42 ± 3 and 43 ± 4 μM, respectively. The NADH oxidase was a heterodimeric flavoprotein with two subunits with molecular masses of 54 kDa and 46 kDa. Its gene sequences were identified, and the enzyme might represent a new type of NADH oxidase in anaerobes. An NADH-dependent peroxidase with a specific activity of 0.1 U/mg was also present in the cell extract of T. maritima.

Discovery and characterization of a novel ATP/polyphosphate xylulokinase from a hyperthermophilic bacterium Thermotoga maritima

2013 ◽  
Vol 40 (7) ◽  
pp. 661-669 ◽  
Author(s):  
Julia S. Martín del Campo ◽  
You Chun ◽  
Jae-Eung Kim ◽  
Rodrigo Patiño ◽  
Y.-H. Percival Zhang
Keyword(s):  
Thermotoga Maritima ◽  
Hyperthermophilic Bacterium

Characterization of a thermostable carboxylesterase from the hyperthermophilic bacterium Thermotoga maritima

2007 ◽  
Vol 74 (3) ◽  
pp. 585-591 ◽  
Author(s):  
Satoshi Kakugawa ◽  
Shinya Fushinobu ◽  
Takayoshi Wakagi ◽  
Hirofumi Shoun
Keyword(s):  
Thermotoga Maritima ◽  
Hyperthermophilic Bacterium

Rhizobium etli HrpW is a pectin-degrading enzyme and differs from phytopathogenic homologues in enzymically crucial tryptophan and glycine residues

Microbiology ◽  
2009 ◽  
Vol 155 (9) ◽  
pp. 3045-3054 ◽  
Author(s):  
Maarten Fauvart ◽  
Natalie Verstraeten ◽  
Bruno Dombrecht ◽  
Ruth Venmans ◽  
Serge Beullens ◽  
...  
Keyword(s):  
Pathogenic Bacteria ◽  
Plant Cell Wall ◽  
Biochemical Characterization ◽  
Sequence Data ◽  
Functional Characterization ◽  
Pectate Lyase ◽  
Site Directed Mutagenesis ◽  
Rhizobium Etli ◽  
Lyase Activity

While establishing a nitrogen-fixing symbiosis with leguminous plants, rhizobia are faced with the problem of penetrating the plant cell wall at several stages of the infection process. One of the major components of this barrier is pectin, a heteropolysaccharide composed mainly of galacturonic acid subunits. So far, no enzymes capable of degrading pectin have been isolated from rhizobia. Here, we make an inventory of rhizobial candidate pectinolytic enzymes based on available genome sequence data and present an initial biochemical and functional characterization of a protein selected from this list. Rhizobium etli hrpW is associated with genes encoding a type III secretion system, a macromolecular structure that allows bacteria to directly inject so-called effector proteins into a eukaryotic host's cell cytosol and an essential virulence determinant of many Gram-negative pathogenic bacteria. In contrast to harpin HrpW from phytopathogens, R. etli HrpW possesses pectate lyase activity and is most active on highly methylated substrates. Through comparative sequence analysis, three amino acid residues crucial for the observed enzymic activity were identified: Trp192, Gly212 and Gly213. Their importance was confirmed by site-directed mutagenesis and biochemical characterization of the resulting proteins, with the tryptophan mutant showing no detectable pectate lyase activity and the double-glycine mutant's activity reduced by about 80 %. Surprisingly, despite hrpW expression being induced specifically on the plant root surface, a knockout mutation of the gene does not appear to affect symbiosis with the common bean Phaseolus vulgaris.

Identification and biochemical characterization of threonine dehydratase from the hyperthermophile Thermotoga maritima

Amino Acids ◽  
2021 ◽  
Author(s):  
Tetsuya Miyamoto ◽  
Masumi Katane ◽  
Yasuaki Saitoh ◽  
Masae Sekine ◽  
Kumiko Sakai-Kato ◽  
...  
Keyword(s):  
Biochemical Characterization ◽  
Thermotoga Maritima ◽  
Threonine Dehydratase
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