Browsing by Author "Natarajan, Krishnamurthy"

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Cloning and characterization of 5'-flanking region of oxalate decarboxylase gene from Flammulina velutipes
(Biochemical Society, 2002) Azam, Mohammad; Kesarwani, Meenu; Chakraborty, Subhra; Natarajan, Krishnamurthy; Datta, Asis
The oxalate-degrading enzyme, oxalate decarboxylase (OXDC), was purified and characterized from Flammulina elutipes, a basidiomycetous fungus [Mehta and Datta (1991) J. Biol. Chem. 266, 23548–23553]. The cDNA cloning and analyses revealed that OXDC transcription was induced by oxalic acid. However, in this report, we show that OXDC transcription is induced by low pH, not by oxalate. To understand the regulatory mechanism of OXDC expression, we have cloned and analysed a 580-bp genomic fragment from the 5h-flanking region of the OXDC gene. Sequence analysis showed the presence of several eukaryotic transcription factor binding motifs within the k580 bp of the upstream region. Electrophoretic-mobility-shift assays with partially purified cell extracts revealed specific binding of a factor in acid-induced, but not in uninduced, extracts. Furthermore, DNase I protection assays using the partially purified fraction from oxalic acid-induced extract revealed a footprint of a 13-bp sequence 5hGCGGGGTCGCCGA3h, termed low pH responsive element (LPRE), corresponding to the k287 to k275 bp region of the OXDC promoter. Our results suggest that in F. elutipes cells, activation of OXDC transcription in response to low pH is mediated by the binding of a novel transcription factor through the LPRE site in the OXDC promoter.
N-acetylglucosamine kinase, HXK1 is involved in morphogenetic transition and metabolic gene expression in Candida albicans
(PLOS, 2013) Rao, Kongara Hanumantha; Ghosh, Swagata; Natarajan, Krishnamurthy; Datta, Asis
Candida albicans, a common fungal pathogen which diverged from the baker’s yeast Saccharomyces cerevisiae has the unique ability to utilise N-acetylglucosamine, an amino sugar and exhibits phenotypic differences. It has acquired intricate regulatory mechanisms at different levels in accordance with its life style. N-acetylglucosamine kinase, a component of the N-acetylglucosamine catabolic cascade is an understudied gene since Saccharomyces cerevisiae lacks it. We report HXK1 to act as both positive and negative regulator of transcription of genes involved in maintaining cellular homeostasis. It is involved in repression of hyphal specific genes in addition to metabolic genes. Its regulation of filamentation and GlcNAc metabolism is independent of the known classical regulators like EFG1, CPH1, RAS1, TPK2 or TUP1. Moreover, Hxk1-GFP is localised to cytoplasm, nucleus and mitochondria in a condition specific manner. By employing two-step affinity purification, we report the interaction of HXK1 with SIR2 under filamentation inducing conditions. Our work highlights a novel regulatory mechanism involved in filamentation repression and attempts to decipher the GlcNAc catabolic regulatory cascade in eukaryotes.
A secretion signal is present in the Collybia velutipes oxalate decatboxylase gene
(Elsevier B.V., 2001) Azam, Mohammad; Kesarwani, Meenu; Natarajan, Krishnamurthy; Datta, Asis
The oxalate decarboxylase (OXDC) gene from Collybia velutipes is overexpressed as an active form in Schizosaccharomyces pombe. The recombinant enzyme shows similar pH optima and stability, while substrate kinetic analysis shows a ninefold decrease in K m value with respect to native OXDC. Most of the expressed protein was present in periplasm and remained firmly bound to cell-wall materials. However, 20% of enzyme expressed was secreted out into the medium suggesting the presence of a secretion signal (C. velutipes) in the oxalate decarboxylase gene. This secretion signal is associated with the N-terminal of OXDC as is evident by secretion of nonsecretory genes AmA1 and Beta-galactosidase. An expression vector using this signal is constructed for expression and secretion of heterologous proteins in S. pombe.