Papers by Aniruddha Raychaudhuri
Alanine aminotransferase: amino acid metabolism in higher plants
Amino acids in higher plants
Alanine aminotransferase (AlaAT, EC 2.6.1.2) catalyses the reversible transfer of an amino group ... more Alanine aminotransferase (AlaAT, EC 2.6.1.2) catalyses the reversible transfer of an amino group from alanine (Ala) to 2-oxoglutarate to form pyruvate and glutamate. AlaAT is a pyridoxal phosphate-dependent enzyme that serves as the main route of alanine biosynthesis and degradation. This chapter provides a comprehensive review of the biochemical characteristics of AlaAT and provides insight into the physiological and metabolic functions of the enzyme as reported from several elegant studies across the world. The diversity in cellular and sub-cellular localizations of multiple AlaAT homologues indicates their involvement in multifaceted metabolic processes, including amino acid biosynthesis and primary carbon metabolism. The enzyme is fairly ubiquitous, occurring across different phyla - fungi, algae, mycorrhizae and higher plants, and existing as various isoforms. Detailed kinetic characterization of the enzyme from several laboratories provides interesting details about its pletho...
3. Histidine biosynthesis pathway: A test case for engineering plant metabolism and growth
Biochemical understanding of the factors influencing metabolic pathways, such as the kinetics of ... more Biochemical understanding of the factors influencing metabolic pathways, such as the kinetics of the constituent enzymes and their means of regulation is essential to identify targets for successful pathway engineering. The regulation of these pathways is often complex and influenced by several elements such as rate- limiting and disequilibrium steps, homeostasis, alternate 'bypasses', metabolic compartmentalization, posttranslational modifications, etc. In many cases, ascertaining the precise nodes optimal for pathway engineering is a challenge. Even mathematical modeling that relies on biochemical data for seemingly linear pathways can be challenging due to many of the influencing factors described above. Histidine (His) biosynthesis exemplifies a metabolic pathway regulated by several biochemical factors (Fersht, 1999). This pathway is ubiquitous across bacteria, yeast and plants although the details differ considerably even among closely related organisms. In this chapte...
Agricultural & Environmental Letters, 2020
Drought stress is a prevalent environmental factor that results in significant yield losses for m... more Drought stress is a prevalent environmental factor that results in significant yield losses for maize (Zea mays L.). Genetic improvements through modern breeding methods and the introduction of biotechnology‐derived traits have been used to improve tolerance to drought stress. Conducting yield efficacy trials in the field for plants that exhibit tolerance to abiotic stress poses several challenges, including the inability to control multiple unpredictable environmental stressors. Controlled environment conditions were tested to evaluate yield parameters commonly observed in the field for MON 87460 maize expressing the cold shock protein B (CspB) protein. Maize plants subjected to water‐limitation treatment in the controlled environment exhibited phenotypic characteristics of drought stress consistent with those observed under field conditions. MON 87460 exhibited significant relative increases in ear length, kernel number, and grain weight following water‐limitation treatment. These...
Plant Physiology, 1997
L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) from cyanobacterial (Spirulina platensis), algal... more L-myo-inositol 1-phosphate synthase (EC 5.5.1.4) from cyanobacterial (Spirulina platensis), algal (Euglena gracilis), and higher plant (Oryza sativa, Vigna radiata) sources was purified to electrophoretic homogeneity, biochemically characterized, and compared. Both chloroplastic and cytosolic forms of the enzyme were detected in E. gracilis, O. sativa, and V. radiata, whereas only the cytosolic form was detected in streptomycin-bleached or chloroplastic mutants of E. gracilis and in S. platensis. Both the chloroplastic and cytosolic forms from different sources could be purified following the same three-step chromatographic protocol. L-myo-inositol 1-phosphate synthases purified from these different sources do not differ significantly with respect to biochemical and kinetic parameters except for the molecular mass of the chloroplastic and cytosolic native holoenzymes, which appear to be homotetrameric and homotrimeric associations of their constituent subunits, respectively. Monoval...
Molecular Genetic Analysis of Constitutively Photomorphogenic Mutants of Arabidopsis
Signal Transduction in Plants, 2001
Molecular Genetic Analysis of Constitutively Photomorphogenic Mutants of Arabidopsis JITENDRA P. ... more Molecular Genetic Analysis of Constitutively Photomorphogenic Mutants of Arabidopsis JITENDRA P. KHURANA, AKHILESH K. TYAGI, PARAMJIT KHURANA, ANJU KOCHHAR, PRADEEP K. JAIN, ANIRUDDHA RAYCHAUDHURJ, REKHA CHAWLA, ARVIND K. BHARTI, ...
Salinity-induced enhancement of L-myo-inositol 1-phosphate synthase in rice (Oryza sativa L.)
Plant, Cell and Environment, 1996
ABSTRACT
Plant Physiology, 2002
The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned ... more The gene encoding hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme, has been cloned from soybean (Glycine max). The gene encodes a protein that is 560 amino acids in length and contains a 31-amino acid signal sequence at the N terminus that is not present in the mature protein. The presence of two SKL motifs near the C terminus suggests that the protein resides in the peroxisome. This expectation is borne out by results from immunogold electron microscopy, which revealed that hydroxyisourate hydrolase was localized in the peroxisomes of uninfected root nodules. The gene encoding hydroxyisourate hydrolase was expressed inEscherichia coli, and soluble, catalytically active enzyme was purified to homogeneity. Sequence analysis revealed considerable homology with members of the β-glucosidase family of enzymes. Two glutamate residues, E199 and E408, align with the conserved glutamates that play catalytic roles in the β-glucosidases. However, the other residues that have been...
Plant Molecular Biology, 2006
Based upon the phenotype of young, darkgrown seedlings, a cytokinin-resistant mutant, cnr1, has b... more Based upon the phenotype of young, darkgrown seedlings, a cytokinin-resistant mutant, cnr1, has been isolated, which displays altered cytokinin-and auxin-induced responses. The mutant seedlings possess short hypocotyls and open apical hooks (in dark), and display agravitropism, hyponastic cotyledons, reduced shoot growth, compact rosettes and short roots with increased adventitious branching and reduced number of root hairs. A number of these features invariably depend upon auxin/cytokinin ratio but the cnr1 mutant retains normal sensitivity towards auxin as well as auxin polar transport inhibitor, TIBA, although upregulation of primary auxin-responsive Aux/IAA genes is reduced. The mutant shows resistance towards cytokinin in hypocotyl/ root growth inhibition assays, displays reduced regeneration in tissue cultures (cytokinin response) and decreased sensitivity to cytokinin for anthocyanin accumulation. It is thus conceivable that due to reduced sensitivity to cytokinin, the cnr1 mutant also shows altered auxin response. Surprisingly, the mutant retains normal sensitivity to cytokinin for induction of primary response genes, the type-A Arabidopsis response regulators, although the basal level of their expression was considerably reduced as compared to the wild-type. The zeatin and zeatin riboside levels, as estimated by HPLC, and the cytokinin oxidase activity were comparable in the cnr1 mutant and the wild-type. The hypersensitivity to red light (in hypocotyl growth inhibition assay), partial photomorphogenesis in dark, and hypersensitivity to sugars, are some other features displayed by the cnr1 mutant. The lesion in the cnr1 mutant has been mapped to the top of chromosome 1 where no other previously known cytokinin-resistant mutant has been mapped, indicating that the cnr1 mutant defines a novel locus involved in hormone, light and sugar signalling.
Biochemistry, 2005
The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine ... more The enzyme RhlI catalyzes the formation of N-butyrylhomoserine lactone from S-adenosylmethionine and N-butyrylacyl carrier protein. N-Butyrylhomoserine lactone serves as a quorum-sensing signal molecule in Pseudomonas aeruginosa, and is implicated in the regulation of many processes involved in bacterial virulence and infectivity. The P. aeruginosa genome contains three genes encoding acyl carrier proteins. We have cloned all three genes, expressed the acyl carrier proteins, and characterized each as a substrate for RhlI. A continuous, spectrophotometric assay was developed to facilitate kinetic and mechanistic studies of RhlI. Acp1, which has not been characterized previously, was a good substrate for RhlI, with a K m of 7 µM; the reaction proceeded with a k cat value of 0.35 s -1 . AcpP, which supports fatty acid biosynthesis, was also a good substrate in the RhlI reaction, where k cat was 0.46 s -1 , and the K m for AcpP was 6 µM. The third acyl carrier protein, Acp3, was a poor substrate for RhlI, with a K m of 280 µM; k cat was 0.03 s -1 . Taken together with microarray data from the literature which show that expression of the gene encoding Acp1 is under the control of the quorum-sensing system, our data suggest that Acp1 is likely to be the substrate for RhlI in vivo. Isotope labeling studies were conducted to investigate the chemical mechanism of the RhlI-catalyzed lactonization reaction. Solvent deuterons were not incorporated into product, which implicates a direct attack mechanism in which the carboxylate oxygen of the presumptive N-butyryl-SAM intermediate attacks the methylene carbon adjacent to the sulfonium ion. Alternative mechanisms, in which N-butyrylvinylglycine is formed via elimination of methylthioadenosine, were ruled out on the basis of the observation that RhlI failed to convert authentic N-butyrylvinylglycine to N-butyryl-L-homoserine lactone.
Reactivity and Reaction Order in Acylhomoserine Lactone Formation by Pseudomonas aeruginosa RhlI
Biochemistry, 2008
The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient... more The formation of N-butyrylhomoserine lactone catalyzed by RhlI has been investigated by transient-state kinetic methods. A single intermediate, assigned to N-butyryl- S-adenosylmethionine, was observed. Under single-turnover conditions, the intermediate formed with a rate constant of 4.0 +/- 0.2 s (-1) and decayed with a rate constant of 3.7 +/- 0.2 s (-1). No other intermediates were detected, demonstrating that the RhlI reaction proceeds via acylation of S-adenosylmethionine, followed by lactonization. S-Adenosylhomocysteine acted as a pseudosubstrate, in that it did not undergo either acylation or lactonization but did induce the deacylation of butyryl-acyl carrier protein. The K m for S-adenosylhomocysteine was approximately 15-fold higher than the K m for S-adenosylmethionine. The reactivities of acylated and unacylated sulfonium ions that were analogues of S-adenosylmethionine were investigated by computational methods. The calculations indicated that acylation of the substrate amino group activated the substrate for lactonization, by allowing the carboxyl group oxygen to approach more closely the methylene carbon to which it adds. This observation provides a satisfying chemical rationale for the order of the individual reactions in the catalytic cycle.
A Familiar Motif in a New Context: the Catalytic Mechanism of Hydroxyisourate Hydrolase
Biochemistry, 2003
Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway... more Hydroxyisourate hydrolase is a recently discovered enzyme that participates in the ureide pathway in soybeans. Its role is to catalyze the hydrolysis of 5-hydroxyisourate, the product of the urate oxidase reaction. There is extensive sequence homology between hydroxyisourate hydrolase and retaining glycosidases; in particular, the conserved active site glutamate residues found in retaining glycosidases are present in hydroxyisourate hydrolase as Glu 199 and Glu 408. However, experimental investigation of their roles, as well as the catalytic mechanism of the enzyme, have been precluded by the instability of 5-hydroxyisourate. Here, we report that diaminouracil serves as a slow, alternative substrate and can be used to investigate catalysis by hydroxyisourate hydrolase. The activity of the E199A protein was reduced 400-fold relative to wild-type, and no activity could be detected with the E408A mutant. Steady-state kinetic studies of the wild-type protein revealed that the pH-dependence of V(max) and V/K describe bell-shaped curves, consistent with the hypothesis that catalysis requires two ionizable groups in opposite protonation states. Addition of 100 mM azide accelerated the reaction catalyzed by the wild-type enzyme 8-fold and the E199A mutant 20-fold but had no effect on the E408A mutant. These data suggest that Glu 408 acts as a nucleophile toward the substrate forming a covalent anhydride intermediate, and Glu 199 facilitates formation of the intermediate by serving as a general acid and then activates water for hydrolysis of the intermediate. Thus, the mechanism of hydroxyisourate hydrolase is strikingly similar to that of retaining glycosidases, even though it catalyzes hydrolysis of an amide bond.
Biochemistry, 2006
Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penult... more Alginate is a major constituent of mature biofilms produced by Pseudomonas aeruginosa. The penultimate step in the biosynthesis of alginate is the conversion of some β-D-mannuronate residues in the polymeric substrate polymannuronan to α-L-guluronate residues in a reaction catalyzed by C5-mannuronan epimerase. Specificity studies conducted with size-fractionated oligomannuronates revealed that the minimal substrate contained 9 monosaccharide residues. The maximum velocity of the reaction increased from 0.0018 s -1 to 0.0218 s -1 as the substrate size increased from 10 to 20 residues, and no additional increase in k cat was observed for substrates up to 100 residues in length. The K m decreased from 80 μM for substrate containing fewer than 15 residues to 4 μM for substrate containing over 100 residues. In contrast to C5-mannuronan epimerases that have been characterized in other bacterial species, P. aeruginosa C5-mannuronan epimerase does not require Ca 2+ for activity, and the Ca 2+ -alginate complex is not a substrate for the enzyme. Analysis of purified, active enzyme by inductively coupled plasma-emission spectroscopy revealed that no metals were present in the protein. The pH dependence of the kinetic parameters revealed that 3 residues on the enzyme which all have a pK a of about 7.6 must be protonated for catalysis to occur. The composition of the polymeric product of the epimerase reaction was analyzed by 1 H-NMR spectroscopy, which revealed that tracts of adjacent guluronate residues were readily formed. The reaction reached an apparent equilibrium when the guluronate composition of the polymer was 75%.
Uploads
Papers by Aniruddha Raychaudhuri