Recombinant tyrosine aminotransferase from Trypanosoma cruzi: structural characterization and site directed mutagenesis of a broad substrate specificity enzyme

Trypanosoma cruzi, the human parasite that causes Chagas disease, contains a functional pentose phosphate pathway, probably essential for protection against oxidative stress and also for R5P (ribose 5-phosphate) production for nucleotide synthesis. The haploid genome of the CL Brener clone of the parasite contains one gene coding for a Type B Rpi (ribose 5-phosphate isomerase), but genes encoding Type A Rpis, most frequent in eukaryotes, seem to be absent. The RpiB enzyme was expressed in Escherichia coli as a poly-His tagged active dimeric protein, which catalyses the reversible isomerization of R5P to Ru5P (ribulose 5-phos-phate) with Km values of 4 mM (R5P) and 1.4 mM (Ru5P).4-Phospho-D-erythronohydroxamic acid, an analogue to the reaction intermediate when the Rpi acts via a mechanism involving the formation of a 1,2-cis-enediol, inhibited the enzyme competi-tively, with an IC50 value of 0.7 mM and a Ki of 1.2 mM. Site-directed mutagenesis allowed the demonstration of a role for His102, but not for His138, in the opening of the ribose furanosic ring. A major role in catalysis was confirmed for Cys69, since the C69A mutant was inactive in both forward and reverse directions of the reaction. The present paper contributes to the know-ledge of the mechanism of the Rpi reaction; in addition, the absence of RpiBs in the genomes of higher animals makes this enzyme a possible target for chemotherapy of Chagas disease.

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Site-directed mutagenesis of AMP-binding residues in adenylate kinase Alteration of substrate specificity

FEBS Letters ◽

10.1016/0014-5793(93)81687-u ◽

1993 ◽

Vol 334 (1) ◽

pp. 86-88 ◽

Cited By ~ 12

Author(s):

Toshihide Okajima ◽

Katsuyuki Tanizawa ◽

Toshio Fukui

Keyword(s):

Substrate Specificity ◽

Adenylate Kinase ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Binding Residues

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Versatile Fungal Polyphenol Oxidase with Chlorophenol Bioremediation Potential: Characterization and Protein Engineering

Applied and Environmental Microbiology ◽

10.1128/aem.01628-18 ◽

2018 ◽

Vol 84 (23) ◽

Cited By ~ 2

Author(s):

Efstratios Nikolaivits ◽

Maria Dimarogona ◽

Ioanna Karagiannaki ◽

Angelina Chalima ◽

Ayelet Fishman ◽

...

Keyword(s):

Substrate Specificity ◽

Fruits And Vegetables ◽

Homology Model ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Gene Encoding ◽

Content Type ◽

Polyphenol Oxidases ◽

Increased Activity ◽

Work Knowledge

ABSTRACTPolyphenol oxidases (PPOs) have been mostly associated with the undesirable postharvest browning in fruits and vegetables and have implications in human melanogenesis. Nonetheless, they are considered useful biocatalysts in the food, pharmaceutical, and cosmetic industries. The aim of the present work was to characterize a novel PPO and explore its potential as a bioremediation agent. A gene encoding an extracellular tyrosinase-like enzyme was amplified from the genome ofThermothelomyces thermophilaand expressed inPichia pastoris. The recombinant enzyme (TtPPO) was purified and biochemically characterized. Its production reached 40 mg/liter, and it appeared to be a glycosylated and N-terminally processed protein.TtPPO showed broad substrate specificity, as it could oxidize 28/30 compounds tested, including polyphenols, substituted phenols, catechols, and methoxyphenols. Its optimum temperature was 65°C, with a half-life of 18.3 h at 50°C, while its optimum pH was 7.5. The homology model ofTtPPO was constructed, and site-directed mutagenesis was performed in order to increase its activity on mono- and dichlorophenols (di-CPs). The G292N/Y296V variant ofTtPPO 5.3-fold increased activity on 3,5-dichlorophenol (3,5-diCP) compared to the wild type.IMPORTANCEA novel fungal PPO was heterologously expressed and biochemically characterized. Construction of single and double mutants led to the generation of variants with altered specificity against CPs. Through this work, knowledge is gained regarding the effect of mutations on the substrate specificity of PPOs. This work also demonstrates that more potent biocatalysts for the bioremediation of harmful CPs can be developed by applying site-directed mutagenesis.

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Site-Directed Mutagenesis of the Redox-Active Cysteines of Trypanosoma cruzi Trypanothione Reductase

European Journal of Biochemistry ◽

10.1111/j.1432-1033.1995.tb20319.x ◽

1995 ◽

Vol 228 (3) ◽

pp. 745-752 ◽

Cited By ~ 55

Author(s):

Adolfo Borges ◽

Mark L. Cunningham ◽

Jorge Tovar ◽

Alan H. Fairlamb

Keyword(s):

Trypanosoma Cruzi ◽

Site Directed Mutagenesis ◽

Directed Mutagenesis ◽

Trypanothione Reductase ◽

Redox Active

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Functional and structural characterization of IdnL7, an adenylation enzyme involved in incednine biosynthesis

Acta Crystallographica Section F Structural Biology Communications ◽

10.1107/s2053230x19002863 ◽

2019 ◽

Vol 75 (4) ◽

pp. 299-306

Author(s):

Jolanta Cieślak ◽

Akimasa Miyanaga ◽

Makoto Takaishi ◽

Fumitaka Kudo ◽

Tadashi Eguchi

Keyword(s):

Amino Acids ◽

Substrate Specificity ◽

Natural Product ◽

Structural Characterization ◽

Biochemical Analysis ◽

Natural Product Biosynthesis ◽

Broad Substrate Specificity ◽

Selective Incorporation ◽

Polyketide Antibiotic

Adenylation enzymes play an important role in the selective incorporation of the cognate carboxylate substrates in natural product biosynthesis. Here, the biochemical and structural characterization of the adenylation enzyme IdnL7, which is involved in the biosynthesis of the macrolactam polyketide antibiotic incednine, is reported. Biochemical analysis showed that IdnL7 selects and activates several small amino acids. The structure of IdnL7 in complex with an L-alanyl-adenylate intermediate mimic, 5′-O-[N-(L-alanyl)sulfamoyl]adenosine, was determined at 2.1 Å resolution. The structure of IdnL7 explains the broad substrate specificity of IdnL7 towards small L-amino acids.

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