F420H2-dependent enzymes reduce a wide range of substrates that are otherwise recalcitrant to enzyme-catalyzed reduction, and their potential for applications in biocatalysis has attracted increasingly attention. Thermobifida fusca is a moderately thermophilic bacterium and holds high biocatalytic potential as a source for several highly thermostable enzymes. We report here on the isolation and characterization of a thermostable F420:NADPH oxidoreductase (Tfu-FNO) from T. fusca, being the first F420-dependent enzyme described from this bacterium. Tfu-FNO was heterologously expressed in Escherichia coli, yielding up to 200 mg recombinant enzyme per liter of culture. We found that Tfu-FNO is highly thermostable, reaching its highest activity at 65 °C and that Tfu-FNO is likely to act in vivo as an F420 reductase at the expense of NADPH, similar to its counterpart in Streptomyces griseus. We obtained the crystal structure of FNO in complex with NADP+ at 1.8 Å resolution, providing the first bacterial FNO structure. The overall architecture and NADP+-binding site of Tfu-FNO were highly similar to those of the Archaeoglobus fulgidus FNO (Af-FNO). The active site is located in a hydrophobic pocket between an N-terminal dinucleotide-binding domain and a smaller C-terminal do-main. Residues interacting with the 2′-phosphate of NADP+ were probed by targeted mutagenesis, indicating that Thr28, Ser50, Arg51, and Arg55 are important for discriminating between NADP+ and NAD+. Interestingly, a T28A mutant increased the kinetic efficiency more than three-fold as compared with the wild-type enzyme when NADH is the substrate. The biochemical and structural data presented here provide crucial insights into the molecular recognition of the two cofactors, F420 and NAD(P)H by FNO.
from #AlexandrosSfakianakis via Alexandros G.Sfakianakis on Inoreader http://ift.tt/2pBMljK
via IFTTT
Εγγραφή σε:
Σχόλια ανάρτησης (Atom)
Δημοφιλείς αναρτήσεις
-
Publication date: Available online 4 January 2018 Source: European Journal of Radiology Author(s): Peiyao Zhang, Jing Wang, Qin Xu, Zhen...
-
BACKGROUND AND PURPOSE: Lesion load is a common biomarker in multiple sclerosis, yet it has historically shown modest association with cl...
-
Does CBD Oil Lower Blood Pressure? This article was originally published at SundayScaries." Madeline Taylor POSTED ON January 13, 20...
-
Despite widespread journal endorsement of reporting guidelines, the poor reproducibility of preclinical research is increasingly under debat...
-
The benefit of the breastfeeding has been well-established. In comparison to partial breast feeding, exclusive breastfeeding has even more b...
-
A report of the Cold Spring Harbor Laboratory/Wellcome Trust Meeting on Engineering Principles in Biology, Cambridge, UK, 14-16 October 2009...
-
Abstract We report here a microscopic tight-binding theoretical study of the dynamic dielectric response of graphene-on-polarizable substr...
-
Volumetry measurement of lung nodules is gaining clinical acceptance but still... Read more on AuntMinnieEurope.com Related Reading: Ul...
-
Background Hyperthyroidism is associated with increased thrombotic risk. As contact system activation through formation of neutrophil extrac...
Δεν υπάρχουν σχόλια:
Δημοσίευση σχολίου