A Study on the Interaction of Rhodamine B with Methylthioadenosine Phosphorylase Protein Sourced from an Antarctic Soil Metagenomic Library

نویسندگان

  • Paulina Bartasun
  • Hubert Cieśliński
  • Anna Bujacz
  • Anna Wierzbicka-Woś
  • Józef Kur
چکیده

The presented study examines the phenomenon of the fluorescence under UV light excitation (312 nm) of E. coli cells expressing a novel metagenomic-derived putative methylthioadenosine phosphorylase gene, called rsfp, grown on LB agar supplemented with a fluorescent dye rhodamine B. For this purpose, an rsfp gene was cloned and expressed in an LMG194 E. coli strain using an arabinose promoter. The resulting RSFP protein was purified and its UV-VIS absorbance spectrum and emission spectrum were assayed. Simultaneously, the same spectroscopic studies were carried out for rhodamine B in the absence or presence of RSFP protein or native E. coli proteins, respectively. The results of the spectroscopic studies suggested that the fluorescence of E. coli cells expressing rsfp gene under UV illumination is due to the interaction of rhodamine B molecules with the RSFP protein. Finally, this interaction was proved by a crystallographic study and then by site-directed mutagenesis of rsfp gene sequence. The crystal structures of RSFP apo form (1.98 Å) and complex RSFP/RB (1.90 Å) show a trimer of RSFP molecules located on the crystallographic six fold screw axis. The RSFP complex with rhodamine B revealed the binding site for RB, in the pocket located on the interface between symmetry related monomers.

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

منابع مشابه

An examination of an Antarctic soil metagenomic-derivate putative methylthioadenosine phosphorylase gene as a novel reporter gene for promoter trapping.

The reporter genes are widely used in promotertrapping vectors for easy and rapid identifi cation of the putative promoter sequences in cloned DNA fragments and in some cases to measure the promoter strength. In this aim, the reporter genes such as a gfp gene from Aequorea victoria encoding Green Fluorescent Protein (Miller et al., 2000; Pothier et al., 2007; Rasko et al., 2007), a lacZ gene en...

متن کامل

Identification and molecular modeling of a novel lipase from an Antarctic soil metagenomic library.

In this work, we present the construction of a metagenomic library in Escherichia coli using pUC19 vector and environmental DNA directly isolated from Antarctic topsoil and screened for lipolytic enzymes. Screening on agar supplemented with olive oil and rhodamine B revealed one clone with lipolytic activity (Lip1) out of 1000 E. coli clones. This clone harbored a plasmid, pLip1, which has an i...

متن کامل

Metagenomic Analysis of a Southern Maritime Antarctic Soil

Our current understanding of Antarctic soils is derived from direct culture on selective media, biodiversity studies based on clone library construction and analysis, quantitative PCR amplification of specific gene sequences and the application of generic microarrays for microbial community analysis. Here, we investigated the biodiversity and functional potential of a soil community at Mars Oas...

متن کامل

Effective Parameters on Removal of Rhodamine B from Colored Wastewater by Nano polyaniline/Sawdust Composite

Background and purpose: Contamination of groundwater with colored and toxic wastewater causes many environmental problems and has adverse effects on human health. This study aimed at coating a nano polyaniline polymer layer on the surface of sawdust and preparing polyaniline sawdust composite (Pan/SD) via chemical polymerization of aniline monomer onto sawdust in aqueous solutions and removal o...

متن کامل

Identification of a novel alkaliphilic esterase active at low temperatures by screening a metagenomic library from antarctic desert soil.

A novel esterase was identified through functional screening of a metagenomic library in Escherichia coli obtained from Antarctic desert soil. The 297-amino-acid sequence had only low (<29%) similarity to a putative esterase from Burkholderia xenovorans. The enzyme was active over a temperature range of 7 to 54 degrees C and at alkaline pH levels and is a potential candidate for industrial appl...

متن کامل

ذخیره در منابع من


  با ذخیره ی این منبع در منابع من، دسترسی به آن را برای استفاده های بعدی آسان تر کنید

برای دانلود متن کامل این مقاله و بیش از 32 میلیون مقاله دیگر ابتدا ثبت نام کنید

ثبت نام

اگر عضو سایت هستید لطفا وارد حساب کاربری خود شوید

عنوان ژورنال:

دوره 8  شماره 

صفحات  -

تاریخ انتشار 2013