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NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features

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ABSTRACT

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

No MeSH data available.


Fluorescence spectra of 0.10 mg/mL AlDHPyr1147 at room temperature: AlDHPyr1147 as isolated (black line), the same enzyme after heating up to 85°C and subsequent cooling to room temperature (red line). The excitation is at 297 and 330 nm (in the inset).
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fig2: Fluorescence spectra of 0.10 mg/mL AlDHPyr1147 at room temperature: AlDHPyr1147 as isolated (black line), the same enzyme after heating up to 85°C and subsequent cooling to room temperature (red line). The excitation is at 297 and 330 nm (in the inset).

Mentions: Figure 2 presents the tryptophan fluorescence spectrum of AlDHPyr1147 as isolated. At an excitation wavelength of 297 nm, the fluorescence spectrum shows two peaks with maxima at 332 (tryptophan fluorescence) and 430 nm (enzyme-bound NADPH fluorescence). At an excitation wavelength of 330 nm, the spectrum shows one peak with a maximum at 430 nm (the inset) corresponding to the enzyme-bound NADPH fluorescence [36]. The heating of the enzyme to 85°С followed by cooling leads to the disappearance of the peak at 430 nm under excitation both at 297 and at 330 nm; that is, the coenzyme dissociates from the enzyme, resulting in the conversion of AlDHPyr1147 to the apo form. The spectrophotometric analysis of low-molecular-weight components after the separation of the heated protein using Amicon Ultra centrifugal filter devices (Millipore) demonstrated the presence of both NADP+ and NADPH in the filtrate. The concentrations of both forms of the coenzyme were estimated using the extinction coefficients of 1.8 × 104 M−1 cm−1 (NADP+ at 260 nm), 1.5 × 104 M−1 cm−1 (NADPH at 260 nm), and 6.22 × 103 M−1 cm−1 (NADPH at 340 nm) [10]. The percentage of NADPH in the filtrate was 30%. It should be noted that the dialysis against 1 M NaCl for 4 days led only to the partial removal of the coenzyme (based on the fluorescence spectra). Thus, AlDHPyr1147 is isolated in complex with coenzyme.


NADP-Dependent Aldehyde Dehydrogenase from Archaeon Pyrobaculum sp.1860 : Structural and Functional Features
Fluorescence spectra of 0.10 mg/mL AlDHPyr1147 at room temperature: AlDHPyr1147 as isolated (black line), the same enzyme after heating up to 85°C and subsequent cooling to room temperature (red line). The excitation is at 297 and 330 nm (in the inset).
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Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC5121451&req=5

fig2: Fluorescence spectra of 0.10 mg/mL AlDHPyr1147 at room temperature: AlDHPyr1147 as isolated (black line), the same enzyme after heating up to 85°C and subsequent cooling to room temperature (red line). The excitation is at 297 and 330 nm (in the inset).
Mentions: Figure 2 presents the tryptophan fluorescence spectrum of AlDHPyr1147 as isolated. At an excitation wavelength of 297 nm, the fluorescence spectrum shows two peaks with maxima at 332 (tryptophan fluorescence) and 430 nm (enzyme-bound NADPH fluorescence). At an excitation wavelength of 330 nm, the spectrum shows one peak with a maximum at 430 nm (the inset) corresponding to the enzyme-bound NADPH fluorescence [36]. The heating of the enzyme to 85°С followed by cooling leads to the disappearance of the peak at 430 nm under excitation both at 297 and at 330 nm; that is, the coenzyme dissociates from the enzyme, resulting in the conversion of AlDHPyr1147 to the apo form. The spectrophotometric analysis of low-molecular-weight components after the separation of the heated protein using Amicon Ultra centrifugal filter devices (Millipore) demonstrated the presence of both NADP+ and NADPH in the filtrate. The concentrations of both forms of the coenzyme were estimated using the extinction coefficients of 1.8 × 104 M−1 cm−1 (NADP+ at 260 nm), 1.5 × 104 M−1 cm−1 (NADPH at 260 nm), and 6.22 × 103 M−1 cm−1 (NADPH at 340 nm) [10]. The percentage of NADPH in the filtrate was 30%. It should be noted that the dialysis against 1 M NaCl for 4 days led only to the partial removal of the coenzyme (based on the fluorescence spectra). Thus, AlDHPyr1147 is isolated in complex with coenzyme.

View Article: PubMed Central - PubMed

ABSTRACT

We present the functional and structural characterization of the first archaeal thermostable NADP-dependent aldehyde dehydrogenase AlDHPyr1147. In vitro, AlDHPyr1147 catalyzes the irreversible oxidation of short aliphatic aldehydes at 60–85°С, and the affinity of AlDHPyr1147 to the NADP+ at 60°С is comparable to that for mesophilic analogues at 25°С. We determined the structures of the apo form of AlDHPyr1147 (3.04 Å resolution), three binary complexes with the coenzyme (1.90, 2.06, and 2.19 Å), and the ternary complex with the coenzyme and isobutyraldehyde as a substrate (2.66 Å). The nicotinamide moiety of the coenzyme is disordered in two binary complexes, while it is ordered in the ternary complex, as well as in the binary complex obtained after additional soaking with the substrate. AlDHPyr1147 structures demonstrate the strengthening of the dimeric contact (as compared with the analogues) and the concerted conformational flexibility of catalytic Cys287 and Glu253, as well as Leu254 and the nicotinamide moiety of the coenzyme. A comparison of the active sites of AlDHPyr1147 and dehydrogenases characterized earlier suggests that proton relay systems, which were previously proposed for dehydrogenases of this family, are blocked in AlDHPyr1147, and the proton release in the latter can occur through the substrate channel.

No MeSH data available.