Anand Bachhawat

• Indian Institute of Science Education and Research Mohali

Gamma-Glu dipeptides are ubiquitous in nature, and yet their metabolism and transport are poorly understood. Here we investigate this using the dipeptide gamma-Glu-met in Saccharomyces cerevisiae. gamma-Glu-met was efficiently utilized by S. cerevisiae and its degradation was dependent on both the glutathione degrading cytosolic Dug2p/Dug3p complex...

The cytosolic glutathione-degrading enzyme, ChaC1, is highly upregulated in several cancers, with the upregulation correlating to poor prognosis. The ability to inhibit ChaC1 thus becomes important in pathophysiological situations where elevated glutathione levels would be beneficial. As no inhibitors of ChaC1 are known, in this study we have focus...

Saccharomyces cerevisiae has been demonstrated to be an excellent platform for the multi-fragment assembly of large DNA constructs through its powerful homologous recombination ability. These assemblies have invariably used the stable centromeric single copy vectors. However, many applications of these assembled genomes would benefit from assembly...

Isoprenoid biosynthesis has a significant requirement for the co‐factor NADPH. Thus, increasing NADPH levels for enhancing isoprenoid yields in synthetic biology is critical. Previous efforts have focused on diverting flux into the pentose phosphate pathway or overproducing enzymes that generate NADPH. In this study, we instead focused on increasin...

The discovery of enzyme deficiencies in lysosomal storage disorders began with two discoveries made in 1963. One of these was made by a Belgian scientist, Henri-Gery Hers, who discovered that in Pompe’s disease there was a deficiency in α-glucosidase. The other was made by an international collaboration involving an American neurologist, James Aust...

Cystinosis is an autosomally inherited rare genetic disorder in which cystine accumulates in the lysosome. The defect arises from a mutation in the lysosomal efflux pump, cystinosin (or CTNS). Despite the disease being known for more than a century, research, diagnosis, and treatment in India have been very minimal. In recent years, however, some r...

Copper plays an underlying role in Host-Leishmania relationship where both parties perturb copper homeostasis to gain advantage over the other.

The glutathione degrading enzyme ChaC1 is highly upregulated in several cancers and viral infections making it a potential pharmacological target for cancer therapy. As an enzyme, however, ChaC1 has a relatively high Km (~2mM) towards its natural substrate, and therefore finding its inhibitors becomes very difficult. Given this limitation, a carefu...

Despite the availability of different antifungal drugs in the market, their overall usefulness remains questionable due to the relatively high toxic profiles exerted by them in many cases. In addition, the emergence of drug resistance against these antifungal agents is a matter of concern. Thus, it becomes imperative to explore innovative drug-deli...

The difficulty in producing multi-carbon and thus high-value chemicals from CO2 is one of the key challenges of microbial electrosynthesis (MES) and other CO2 utilization technologies. Here, we demonstrate a two-stage bioproduction approach to produce terpenoids (>C20) and yeast biomass from CO2 by linking MES and yeast cultivation approaches. In t...

Copper is pivotal for all life forms; however, in excess, it is toxic. Hosts utilize the toxic properties of copper against various pathogenic invasions. Both free-living and intracellular forms of Leishmania exhibit appreciable tolerance towards copper stress. Deciphering the copper-stress evasion mechanism led us to characterize the hitherto Copp...

Copper is essential for all life forms; however, in excess, it becomes toxic. Toxic properties of copper are known to be utilized by host species against various pathogenic invasions. Leishmania, in both free-living and intracellular forms, exhibits appreciable tolerance towards copper-stress. While determining the mechanism of copper-stress evasio...

Copper is essential for all life forms; however in excess it is extremely toxic. Toxic properties of copper are utilized by hosts against various pathogenic invasions. Leishmania, in its both free-living and intracellular forms was found to exhibit appreciable tolerance towards copper-stress. To determine the mechanism of copper-stress evasion empl...

The adenine biosynthetic mutants ade1 and ade2 of Saccharomyces cerevisiae accumulate a characteristic red pigment in their vacuoles under adenine limiting conditions. This red pigmentation phenotype, widely used in a variety of genetic screens and assays, is the end product of a glutathione-mediated detoxification pathway, where the glutathione co...

Methylenetetrahydrofolate reductase (MTHFR) links the folate cycle to the methionine cycle in one-carbon metabolism.The enzyme is known to be allosterically inhibited by S-adenosyl methionine (SAM) for decades, but the importance of this regulatory control to one carbon metabolism has never been adequately understood. To shed light on this issue, w...

Heart failure results from the heart's inability to carryout ventricular contraction and relaxation , and has now become a worldwide problem. During the onset of heart failure, several signatures are observed in cardiomyocytes that includes fetal reprogramming of gene expression where adult genes are repressed and fetal genes turned on, endoplas-mi...

Undergraduate laboratory courses, owing to their larger sizes and shorter time slots, are often conducted in highly structured modes. However, this approach is known to interfere with students' engagement in the experiments. To enhance students' engagement, we propose an alternative mode of running laboratory courses by creating some “disorder” in...

NADPH is an important cofactor in the cell. In addition to its role in the biosynthesis of critical metabolites, it plays crucial roles in the regeneration of the reduced forms of glutathione, thioredoxins and peroxiredoxins. The enzymes and pathways that regulate NADPH are thus extremely important to understand, and yet are only partially understo...

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Calcium signaling is essential for embryonic development but the signals upstream of calcium are only partially understood. Here, we investigate the role of the intracellular glutathione redox potential in calcium signaling using the Chac1 protein of zebrafish. A member of the γ-glutamylcyclotransferase family of enzymes, the zebrafish Chac1 is a g...

The yeast Saccharomyces cerevisiae is one of the preferred hosts for the production of terpenoids through metabolic engineering. A genetic screen to identify novel mutants that can increase the flux in the isoprenoid pathway has been lacking. We present here the method that has led to the development of a carotenoid based visual screen by exploitin...

Background Production of isoprenoids, a large and diverse class of commercially important chemicals, can be achieved through engineering metabolism in microorganisms. Several attempts have been made to reroute metabolic flux towards isoprenoid pathway in yeast. Most approaches have focused on the core isoprenoid pathway as well as on meeting the in...

Glutathione was discovered in 1888, over 125 years ago. Since then, our understanding of various functions and metabolism of this important molecule has grown over these years. But it is only now, in the last decade, that a somewhat complete picture of its metabolism has emerged. Glutathione metabolism has till now been largely depicted and underst...

Cystinosin, a lysosomal transporter is involved in the efflux of cystine from the lysosome to the cytosol. Mutations in the human cystinosin gene (CTNS) cause cystinosis, a recessive autosomal disorder. Studies on cystinosin have been limited by the absence of a robust genetic screen. In the present study we have developed a dual strategy for evalu...

Background: Knowledge of catalytic residues can play an essential role in elucidating mechanistic details of an enzyme. However, experimental identification of catalytic residues is a tedious and time-consuming task, which can be expedited by computational predictions. Despite significant development in active-site prediction methods, one of the r...

Cystine transporters are a clinically important class of transporters found in bacteria, pathogenic fungi and mammalian cells. Despite their significance, very little is known about the mechanism of substrate recognition and transport. We have carried out studies on the plasma membrane Candida glabrata cystine transporter, CgCYN1 a member of the am...

CCH1, the yeast homolog of the pore-forming subunit α1 of the mammalian Voltage-gated calcium channel (VGCC) located on the plasma membrane mediates the redox-dependent influx of calcium. Cch1p is known to undergo both rapid activation (oxidative stress, high pH) and slow activation (ER stress, mating pheromone activation), but the mechanism of act...

Significance: Glutathione degradation has for long been thought to occur only on non-cytosolic pools. This is because there has been only one enzyme known to degrade glutathione (γ-glutamyltranspeptidase), and this localizes to either the plasma membrane (mammals, bacteria) or the vacuolar membrane (yeast, plants) and acts on extracellular or vacu...

Proton gradient acts as the driving force for the transport of many metabolites across fungal and plant plasma membranes. Identifying the mechanism of proton relay is critical for understanding the mechanism of transport mediated by these transporters. We have investigated two strategies for identifying residues critical for proton dependent substr...

Cysteine is an essential requirement in living organisms. However, due to its reactive thiol side chain, elevated levels of intracellular cysteine can be toxic and therefore need to be rapidly eliminated from the cellular milieu. In mammals and many other organisms, excess cysteine is believed to be primarily eliminated by the cysteine dioxygenase...

Glutathione degradation plays an important role in glutathione and redox homeostasis and thus it is imperative to understand the enzymes and the mechanisms involved in glutathione degradation in detail. We describe here ChaC2, a member of the ChaC family of γ-glutamylcyclotransferases, as an enzyme that degrades glutathione in the cytosol of mammal...

Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeast. We show here that glutathione depletion in yeast leads to the activation of two cytoplasmically inward facing channels, the plasma membrane, Cch1p and the vacuolar calcium chan...

3913 MBoC | ARTICLE Glutathione depletion activates the yeast vacuolar transient receptor potential channel, Yvc1p, by reversible glutathionylation of specific cysteines ABSTRACT Glutathione depletion and calcium influx into the cytoplasm are two hallmarks of apoptosis. We have been investigating how glutathione depletion leads to apoptosis in yeas...

The high affinity glutathione transporter Hgt1p, of Saccharomyces cerevisiae belongs to a relatively new and structurally uncharacterized oligopeptide transporter family. To understand the structural features required for interaction with Hgt1p, a quantitative investigation of substrate specificity of Hgt1p was carried out. Hgt1p showed a higher af...

Commercial production of yeast, which exceeds 1.8 million tons per year, owes its success to the optimization of feedstock, energy inputs, physico–chemical parameters, and chemical engineering innovation. The knowledge of the unique adaptive physiology of Saccharomyces cerevisiae to varying aeration rate and glucose concentration in growth medium a...

Inherited 5-oxoprolinase (OPLAH) deficiency is a rare inborn condition characterised by 5-oxoprolinuria. To date, three OPLAH mutations have been described: p.H870Pfs in a homozygous state, which results in a truncated protein, was reported in two siblings, and two heterozygous missense changes, p.S323R and p.V1089I, were independently identified i...

ChaC1 is a mammalian proapoptic protein of unknown function induced during endoplasmic reticulum stress. We show using in vivo studies and novel in vitro assays that the ChaC family of proteins function as γ-glutamyl cyclotransferases acting specifically to degrade glutathione but not other γ-glutamyl peptides. The overexpression of these proteins...

We report the de novo assembled 20.05-Mb draft genome of the red yeast Rhodosporidium toruloides MTCC 457, predicted to encode 5,993 proteins, 4 rRNAs, and 125 tRNAs. Proteins known to be unique to oleaginous fungi are present among the predicted proteins. The genome sequence will be valuable for molecular genetic analysis and manipulation of lipid...

Pyroglutamic acid or 5-oxoproline is the cyclic lactam of glutamic acid. Its presence in living cells has been reported from archaebacteria to humans, and its oc-currence in living cells has been known for over a cen-tury. Despite its almost ubiquitous presence, the role of pyroglutamic acid in living cells is poorly under-stood. Pyroglutamic acid...

We describe a novel plasma membrane cystine transporter, CgCYN1, from Candida glabrata, the first such transporter to be described from yeast and fungi. C. glabrata met15Δ strains, organic sulfur auxotrophs, were observed to utilize cystine as a sulfur source, and this phenotype was exploited in the discovery of CgCYN1. Heterologous expression of C...

Redox pathways play a key role in pathogenesis. Glutathione, a central molecule in redox homeostasis in yeasts, is an essential metabolite, but its requirements can be met either from endogenous biosynthesis or from the extracellular milieu. In this report we have examined the importance of glutathione biosynthesis in two major human opportunistic...

The incidence of fungal infections is increasing worldwide, and the management and treatment of fungal infections has become increasingly important. The antifungals which are currently available target only a few pathways, and their persistent use has resulted not only in increased drug resistance but also in the emergence of newer fungal pathogens...

OXP1/YKL215c, an uncharacterized ORF of Saccharomyces cerevisiae, encodes a functional ATP-dependent 5-oxoprolinase of 1286 amino acids. The yeast 5-oxoprolinase activity was demonstrated in vivo by utilization of 5-oxoproline as a source of glutamate and OTC, a 5-oxoproline sulfur analogue, as a source of sulfur in cells overexpressing OXP1. In vi...

Cystinosis, an inherited disease caused by a defect in the lysosomal cystine transporter (CTNS), is characterized by renal proximal tubular dysfunction. Adenosine triphosphate (ATP) depletion appears to be a key event in the pathophysiology of the disease, even though the manner in which ATP depletion occurs is still a puzzle. We present a model th...

Glutathione (GSH), L-gamma-glutamyl-L-cysteinyl-glycine, is the major low-molecular-weight thiol compound present in almost all eukaryotic cells. GSH degradation proceeds through the gamma-glutamyl cycle that is initiated, in all organisms, by the action of gamma-glutamyl transpeptidase. A novel pathway for the degradation of GSH that requires the...

Frequent outbreaks of Pichia anomala fungaemia in paediatric patients have warranted the development of a rapid identification system for this organism. This study describes a specific PCR-based method targeting the rRNA gene intergenic spacer region 1 (IGS1) for rapid identification of Pichia anomala isolates and characterization at the strain lev...

An important hallmark of biological research is the aspect of ‘comparisons’. As the complete genome sequences of numerous organisms have become available, the emphasis in biology has shifted to comparisons at the genome level. Indeed, the last few years have witnessed an exponential rise in the number of organisms whose complete genome has been seq...

Pichia anomala is an emerging nosocomial pathogen and there is a need for methods that distinguish between different P. anomala strains. In the typing of several clinical as well as non-clinical P. anomala strains, the sequence variation of the internal transcribed spacer (ITS) was found to be inadequate for typing purposes. The intergenic spacer 1...

The ECM38 gene encodes the gamma-glutamyl transpeptidase enzyme, an enzyme involved in glutathione turnover. The enzyme was found to be present in the S288C strain, BY4742, but absent in another widely used strain congenic to S288C, YPH499. Cloning and sequencing the genes from these yeasts indicated the presence of 11 single nucleotide polymorphis...

gamma-Glutamyl transpeptidase (gamma-GT) is the only enzyme known to be responsible for glutathione degradation in living cells. In the present study we provide evidence that the utilization of glutathione can occur in the absence of gamma-GT. When disruptions in the CIS2 gene encoding gamma-GT were created in met15Delta strains, which require orga...

A high affinity glutathione transporter has been identified, cloned, and characterized from the yeastSaccharomyces cerevisiae. This transporter, Hgt1p, represents the first high affinity glutathione transporter to be described from any system so far. The strategy for the identification involved investigating candidate glutathione transporters from...

Membrane IgG H chains turn over considerably more rapidly than secretory Ig H chains in the 18-81 A2 pre-B cell line. This rapid degradation occurs in proteasomes. N-Glycosylated membrane Ig H chains accumulate in the endoplasmic reticulum in the presence of proteasomal inhibitors, suggesting that retrotranslocation and proteasomal degradation of m...

The biosynthesis of inositol requires only two enzymes, inositol-1-phosphate synthase (encoded by INO1) and an inositol monophosphatase, but the regulation of inositol biosynthesis is under multiple controls and is exquisitely regulated. In the budding yeast Saccharomyces cerevisiae, mutations in any of 26 different genes lead to inositol auxotroph...

Mutants of Saccharomyces cerevisiae bearing lesions in the ergosterol biosynthetic pathway exhibit a pleiotropic drug-sensitive phenotype. This has been reported to result from an increased permeability of the membranes of the mutant strains to different drugs. As disruption of the yeast multidrug resistance protein, Pdr5p, results in a similar ple...

We have examined the feasibility and efficiency of PCR-mediated direct gene disruptions in the fission yeast Schizosaccharomyces pombe. In the present study, the S.pombe ura4+ gene was amplified by PCR with oligonucleotides that had short flanking regions (∼40 bp) to the target gene. Using this purified PCR product we were able to disrupt genes in...

Mutants in the adenine biosynthetic pathway of yeasts (ade1 and ade2 of Saccharomyces cerevisiae, ade6 and ade7 of Schizosaccharomyces pombe) accumulate an intense red pigment in their vacuoles when grown under adenine-limiting conditions. The precise events that determine the formation of the pigment are however, still unknown. We have begun a gen...

Silica particles and silica-based resins are widely used for the purification of DNA fragments from agarose gels, from reaction mixtures and for the purification of plasmids (1-5). Several commercial and 'home-made' kits have been described using these matrices (6,7). Despite their wide use, the conditions for DNA elution and the recovery yields ob...

This, the latest in the Sambrook and Tooze Guidebook Series enables non-specialists to rapidly gain access to unfamiliar work surrounding the secretory pathways. Detailing both the established and the experimental, this book outlines how recent work has established a role for the secretory pathway in the regulation of gene expression and cell-cycle...

The highly conserved and ubiquitously expressed mouse gene H < beta > 58, identified through insertional mutagenesis, has been shown to be essential for early postimplantation development in mouse, but the mechanism by which it acts is unknown (Radice et al. 1991; Lee et al. 1992). We report here the identification of a yeast gene related to the H...

Strains bearing the vph2 mutation are defective in vacuolar acidification. The VPH2 gene was isolated from a genomic DNA library by complementation of the zinc-sensitive phenotype of the mutant. Deletion analysis localized the complementing activity to a 1.2 kb DNA fragment. Sequence analysis of this fragment revealed the presence of a single open...

The intracellular fates of membrane and secretory immunoglobulin heavy chains were examined in a pre-B cell line that has switched to the gamma isotype. The membrane form of the heavy chain (gamma m) was rapidly degraded while the secretory form (gamma s) was retained intracellularly in association with BiP. The degradation of gamma m could not be...

ATP:citrate lyase was purified from the oleaginous yeast Rhodotorula gracilis to homogeneity as judged by polyacrylamide gel electrophoresis, using a novel citrate-Sepharose procedure. The enzyme was found to have a molecular weight of 520,000 and consisted of four identical subunits (Mr = 120,000). Two minor low molecular weight bands were observe...

The effect of growth at 42 degrees C on the different components of the siderophore-mediated iron transport that are induced by iron limitation in Azospirillum brasilense was examined. Biosynthesis of the siderophore spirilobactin was strongly inhibited (20-fold) by growth at 42 degrees C, whereas the transport of iron by the ferric-spirilobactin t...

The outer membrane of Azospirillum brasilense was isolated from the total membrane fraction by sucrose density gradient centrifugation and by Sarkosyl extraction; both preparations showed an identical outer membrane protein profile in slab gels after electrophoresis under denaturing conditions. The profile showed a major 42 kDa protein constituting...