Cloning, expression and bioinformatics analysis of ATP sulfurylase from Acidithiobacillus ferrooxidans ATCC 23270 in Escherichia coli |
| |
Authors: | Michael L Jaramillo Michel Abanto Ruth L Quispe Julio Calder��n Lu��s J del Valle Miguel Talledo Pablo Ram��rez |
| |
Institution: | 1Laboratory of Molecular Microbiology and Biotechnology, Faculty of Biological Sciences, Universidad Nacional Mayor de San Marcos, Lima – Peru;2Centre díEnginyeria Biotecnologica i Molecular (CEBIM), Departament díEnginyeria Química, ETSEIB, Universitat Politècnica de Catalunya, Barcelona, Spain |
| |
Abstract: | Molecular studies of enzymes involved in sulfite oxidation in Acidithiobacillus ferrooxidans have not yet been developed, especially
in the ATP sulfurylase (ATPS) of these acidophilus tiobacilli that have importance in biomining. This enzyme synthesizes ATP and
sulfate from adenosine phosphosulfate (APS) and pyrophosphate (PPi), final stage of the sulfite oxidation by these organisms in
order to obtain energy. The atpS gene (1674 bp) encoding the ATPS from Acidithiobacillus ferrooxidans ATCC 23270 was amplified
using PCR, cloned in the pET101-TOPO plasmid, sequenced and expressed in Escherichia coli obtaining a 63.5 kDa ATPS
recombinant protein according to SDS-PAGE analysis. The bioinformatics and phylogenetic analyses determined that the ATPS
from A. ferrooxidans presents ATP sulfurylase (ATS) and APS kinase (ASK) domains similar to ATPS of Aquifex aeolicus, probably of
a more ancestral origin. Enzyme activity towards ATP formation was determined by quantification of ATP formed from E. coli cell
extracts, using a bioluminescence assay based on light emission by the luciferase enzyme. Our results demonstrate that the
recombinant ATP sulfurylase from A. ferrooxidans presents an enzymatic activity for the formation of ATP and sulfate, and possibly
is a bifunctional enzyme due to its high homology to the ASK domain from A. aeolicus and true kinases. |
| |
Keywords: | |
|
|