Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa

Acero-Navarro, Kevin E.; Jiménez-Ramírez, Mariella; Villalobos, Miguel; Vargas-Martínez, Rocío; Perales-Vela, Hugo Virgilio; Velasco-Garcı́a, Roberto

doi:10.1016/j.pep.2017.10.004

Cited by 13 publications

(17 citation statements)

References 39 publications

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“…These differences in native status in the G6PD::6PGL protein from T. vaginalis and G. lamblia could be mainly because both enzymes are different in number (716 aa in G6PD::6PGL from T. vaginalis , and 742 aa in G6PD::6PGL from G. lamblia ), and composition of amino acids, as was previously observed in the alignment of the amino acid sequences of T. vaginalis and G. lamblia , where only a 30% similarity was found between both parasites ( Figure 1 B). In addition, the native status determined for the G6PD::6PGL protein from T. vaginalis is in concordance with the previously reported G6PDs ( Brugia malayi and Pseudomonas aeruginosa ), for which a tetrameric structure was reported [ 32 , 33 ].…”

Section: Resultssupporting

confidence: 90%

“…This result is different with that previously reported for the fused G6PD::6PGL protein from G. lamblia, where the enzyme reached a maximum value at pH 8.75. In this way, the optimal pH determined for TvG6PD::6PGL agrees with that of the other previously purified G6PDs, where pH values of 8.0 were found in G6PDs (in Homo sapiens, B. malayi, buffalo liver, camel liver, dog liver, Taenia crassiceps, Trypanosoma cruzy, Aspergillus Oryzae, Thermotoga maritima, Pseudomonas aeruginosa, and E. coli DH5α [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitysupporting

confidence: 89%

“…Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0. [32][33][34][35][36][37][38][39][40]. Considering these data, the rest of the functional assays for our TvG6PD::6PGL enzyme were performed at a pH of 8.0.…”

Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitymentioning

confidence: 99%

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Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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This report describes a functional and structural analysis of fused glucose-6-phosphate dehydrogenase dehydrogenase-phosphogluconolactonase protein from the protozoan Trichomonas vaginalis (T. vaginalis). The glucose-6-phosphate dehydrogenase (g6pd) gene from T. vaginalis was isolated by PCR and the sequence of the product showed that is fused with 6pgl gene. The fused Tvg6pd::6pgl gene was cloned and overexpressed in a heterologous system. The recombinant protein was purified by affinity chromatography, and the oligomeric state of the TvG6PD::6PGL protein was found as tetramer, with an optimal pH of 8.0. The kinetic parameters for the G6PD domain were determined using glucose-6-phosphate (G6P) and nicotinamide adenine dinucleotide phosphate (NADP+) as substrates. Biochemical assays as the effects of temperature, susceptibility to trypsin digestion, and analysis of hydrochloride of guanidine on protein stability in the presence or absence of NADP+ were performed. These results revealed that the protein becomes more stable in the presence of the NADP+. In addition, we determined the dissociation constant for the binding (Kd) of NADP+ in the protein and suggests the possible structural site in the fused TvG6PD::6PGL protein. Finally, computational modeling studies were performed to obtain an approximation of the structure of TvG6PD::6PGL. The generated model showed differences with the GlG6PD::6PGL protein (even more so with human G6PD) despite both being fused.

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Section: Resultssupporting

confidence: 90%

Section: Effect Of Temperature and Ph On Tvg6pd::6pgl Activitysupporting

confidence: 89%

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Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Morales-Luna

Hernández-Ochoa

Ramírez-Nava

et al. 2020

IJMS

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“…Table 2 displays the kinetic characteristics of purified G6PDHs from various organisms. Compared to these enzymes, except T. maritime G6PDH [38], AoG6PDH has both higher k is 11-fold higher than the Aspergillus enzyme (A. aculeatus G6PDH) [29] and 85% higher than P. aeruginosa G6PDH [39]. Its catalytic efficiency with respect to NADP + was 7-fold higher than that of the human G6PDH [40] and 5-fold higher than that of any of the other G6PDHs.…”

Section: Discussionmentioning

confidence: 99%

Heteroexpression and functional characterization of glucose 6-phosphate dehydrogenase from industrial Aspergillus oryzae

Guo¹,

Han²,

Wu³

et al. 2019

Journal of Microbiology and Biotechnology

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“…While numerous G6PDs from thermophiles and mesophiles are well characterized [4,5,6,7,8], only a small number of G6PDs from psychrophiles are known [9,10,11]. Antonietta et al showed that although G6PDs from cold-adapted fish have a molecular similarity with their mesophilic counterparts, their biochemical characteristics were very different from those of mesophiles, which made the enzymes more appropriate to cold-adapted organisms [11].…”

Section: Introductionmentioning

confidence: 99%

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621

TranNgoc

Pham

Lee

et al. 2019

IJMS

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Glucose 6-phosphate dehydrogenase (G6PD) (EC 1.1.1.363) is a crucial regulatory enzyme in the oxidative pentose phosphate pathway that provides reductive potential in the form of NADPH, as well as carbon skeletons for the synthesis of macromolecules. In this study, we report the cloning, expression, and characterization of G6PD (SpG6PD1) from a lichen-associated psychrophilic bacterium Sphingomonas sp. PAMC 26621. SpG6PD1 was expressed in Escherichia coli as a soluble protein, having optimum activity at pH 7.5–8.5 and 30 °C for NADP+ and 20 °C for NAD+. SpG6PD1 utilized both NADP+ and NAD+, with the preferential utilization of NADP+. A high Km value for glucose 6-phosphate and low activation enthalpy (ΔH‡) compared with the values of mesophilic counterparts indicate the psychrophilic nature of SpG6PD1. Despite the secondary structure of SpG6PD1 being maintained between 4–40 °C, its activity and tertiary structure were better preserved between 4–20 °C. The results of this study indicate that the SpG6PD1 that has a flexible structure is most suited to a psychrophilic bacterium that is adapted to a permanently cold habitat.

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Cloning, overexpression, and purification of glucose-6-phosphate dehydrogenase of Pseudomonas aeruginosa

Cited by 13 publications

References 39 publications

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Characterizing the Fused TvG6PD::6PGL Protein from the Protozoan Trichomonas vaginalis, and Effects of the NADP+ Molecule on Enzyme Stability

Heteroexpression and functional characterization of glucose 6-phosphate dehydrogenase from industrial Aspergillus oryzae

Cloning, Expression, and Characterization of a Psychrophilic Glucose 6-Phosphate Dehydrogenase from Sphingomonas sp. PAMC 26621

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