A calmodulin like EF hand protein positively regulates oxalate decarboxylase expression by interacting with E-box elements of the promoter.

Ayushi Kamthan,Abira Chaudhuri,Mohan Kamthan,Avinash Kumar,Sekhu Ansari,Asis Datta,Pratima Sharma,Sarjeet Singh Thakur

doi:10.1038/srep14578

Abstract

Oxalate decarboxylase (OXDC) enzyme has immense biotechnological applications due to its ability to decompose anti-nutrient oxalic acid. Flammulina velutipes, an edible wood rotting fungus responds to oxalic acid by induction of OXDC to maintain steady levels of pH and oxalate anions outside the fungal hyphae. Here, we report that upon oxalic acid induction, a calmodulin (CaM) like protein-FvCaMLP, interacts with the OXDC promoter to regulate its expression. Electrophoretic mobility shift assay showed that FvCamlp specifically binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter. Moreover, substitutions of amino acids in the EF hand motifs resulted in loss of DNA binding ability of FvCamlp. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript pointing towards positive nature of the regulation. FvCaMLP is different from other known EF hand proteins. It shows sequence similarity to both CaMs and myosin regulatory light chain (Cdc4), but has properties typical of a calmodulin, like binding of 45Ca2+, heat stability and Ca2+ dependent electrophoretic shift. Hence, FvCaMLP can be considered a new addition to the category of unconventional Ca2+ binding transcriptional regulators.

Highlights

Thaliana has been shown to function as a transcriptional regulator by directly binding to a Z-/G-box element located in the promoter of light-responsive genes, and triggering their expression[11]
It was demonstrated that acid-induced cells of F. velutipes contain a low pH responsive factor (LPRF) that binds to a 13 bp sequence (5′ -GCGGGGTCGCCGA-3′ ) in 5′ upstream region of Oxalate decarboxylase (OXDC) gene
Electrophoretic mobility shift assay (EMSA) showed that FvCamlp binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter and amino acid residues from EF hand motifs of the protein are involved in DNA binding

Summary

Introduction

Thaliana has been shown to function as a transcriptional regulator by directly binding to a Z-/G-box element located in the promoter of light-responsive genes, and triggering their expression[11]. OXDC from the wood-rotting fungus F. velutipes was shown to be induced at the transcriptional level by oxalic acid[16] to maintain steady extracellular pH. It was demonstrated that acid-induced cells of F. velutipes contain a LPRF (low pH responsive factor) that binds to a 13 bp sequence (5′ -GCGGGGTCGCCGA-3′ ) in 5′ upstream region of OXDC gene. We show that in presence of oxalic acid, a calmodulin like EF hand protein from F. Electrophoretic mobility shift assay (EMSA) showed that FvCamlp binds to two non-canonical E-box elements (AACGTG) in the OXDC promoter and amino acid residues from EF hand motifs of the protein are involved in DNA binding. F. velutipes mycelia treated with synthetic siRNAs designed against FvCaMLP showed significant reduction in FvCaMLP as well as OXDC transcript, confirming its positive nature of regulation

Methods

Results

Conclusion