Biomedical subjects

G S Prasad

Publications and source records attributed to G S Prasad.

At least 19 recordsLinked to original sources

PubMed2005

Geotrichum silvicola sp. nov., a novel asexual arthroconidial yeast species related to the genus Galactomyces.

Four strains of an asexual arthroconidial yeast species were isolated from Drosophila flies in two Atlantic rain forest sites in Brazil and two strains from oak tasar silkworm larvae (Antheraea proylei) in India. Analysis of the sequences of the D1/D2 large subunit rRNA gene showed that this yeast represented a novel species of the genus Geotrichum, described as Geotrichum silvicola sp. nov. The novel species was related to the ascogenous genus Galactomyces. The closest relatives of Geotrichum silvicola were Galactomyces sp. strain NRRL Y-6418 and Galactomyces geotrichum. The type culture of Geotrichum silvicola is UFMG-354-2T (=CBS 9194T=NRRL Y-27641T).

Animals↗

PubMed2005

Planococcus stackebrandtii sp. nov., isolated from a cold desert of the Himalayas, India.

The taxonomic position of a bacterium isolated from a cold desert of the Himalayas, India, was analysed by using a polyphasic approach. The isolated strain, designated K22-03T, had phenotypic characteristics that matched those of the genus Planococcus and it represents a novel species. The almost-complete 16S rRNA gene sequence (1464 bases) of the novel strain was compared with those of previously studied Planococcus type strains and confirmed that the strain belongs to the genus Planococcus. 16S rRNA gene sequence analysis indicated that strain K22-03T differs from all other species of Planococcus by at least 2.5 %. DNA-DNA hybridization showed that it had low genomic relatedness with Planomicrobium mcmeekinii (MTCC 3704T, 23 %), Planococcus psychrophilus (MTCC 3812T, 61 %), Planococcus antarcticus (MTCC 3854T, 45 %) and Planomicrobium okeanokoites (MTCC 3703T, 51 %), the four species with which it was most closely related based on 16S rRNA gene sequence analysis (97-97.5 % similarity). Therefore, strain K22-03T should be recognized as a novel species, for which the name Planococcus stackebrandtii sp. nov. is proposed. The type strain is K22-03T (=MTCC 6226T=DSM 16419T=JCM 12481T).

Bacterial Typing Techniques↗

PubMed2005

Candida digboiensis sp. nov., a novel anamorphic yeast species from an acidic tar sludge-contaminated oilfield.

Two strains (TERI-6(T) and TERI-7) of a novel yeast species were isolated from acidic tar sludge-contaminated soil samples collected from Digboi Refinery, Assam, India. These two yeast strains were morphologically, physiologically and phylogenetically identical to each other. No sexual reproduction was observed on corn meal, malt, Gorodkowa, YM or V8 agars. Physiologically, the novel isolates were most closely related to Candida blankii, but differed in eight physiological tests. The prominent differences were the ability of the isolates to assimilate melibiose and inulin and their inability to assimilate d-glucuronate, succinate and citrate. Phylogenetic analysis using the D1/D2 variable domain showed that the closest relative of these strains is C. blankii (2.8 % divergence). Other related species are Zygoascus hellenicus and Candida bituminiphila. The isolates differed from C. blankii by 11 base substitutions in the 18S rRNA gene sequence and by 58 base substitutions in the internal transcribed spacer sequences. The physiological, biochemical and molecular data support the contention that strains TERI-6(T) and TERI-7 represent a novel species, for which the name Candida digboiensis sp. nov. is proposed. The type strain is TERI-6(T) (=MTCC 4371(T)=CBS 9800(T)=JCM 12300(T)).

Candida↗

PubMed2005

Aquimonas voraii gen. nov., sp. nov., a novel gammaproteobacterium isolated from a warm spring of Assam, India.

A bacterial strain designated GPTSA 20(T), which was isolated from a warm spring in Assam, India, was characterized by using a polyphasic approach. The cells were Gram-negative, aerobic rods, which could not utilize or produce acid from most of the carbohydrates tested. The predominant fatty acids were C(15:0) iso (25.04%), C(17:1) iso omega9c (19.28%), C(16:0) iso (17.73%) and C(11:0) iso 3-OH (9.34%). The G+C content was 75 mol%. From 16S rRNA gene sequence analysis (1433 nucleotides, continuous stretch), it was confirmed that strain GPTSA 20(T) belonged to the class 'Gammaproteobacteria'. The closest 16S rRNA gene sequence similarity found (98.2%) was with an uncultured bacterium clone, NB-03 (accession no. AB117707), from an autotrophic nitrifying biofilm. Among culturable bacteria, the closest sequence similarities were with Fulvimonas soli (93.0%), Silanimonas lenta (92.8%), Thermomonas hydrothermalis (92.4%), Frateuria aurantia (91.9%), Rhodanobacter lindaniclasticus (91.9%), Thermomonas haemolytica (91.9%) and Pseudoxanthomonas taiwanensis (91.8%); similarities of less than 91.8% were obtained with other members of the class 'Gammaproteobacteria'. From the biochemical, physiological, chemotaxonomic and phylogenetic analysis, it was clear that strain GPTSA 20(T) was quite different from members of known genera of the class 'Gammaproteobacteria'. Therefore, it is proposed that strain GPTSA 20(T) represents a novel species within a new genus, with the name Aquimonas voraii gen. nov., sp. nov. The type strain is GPTSA 20(T) (=MTCC 6713(T)=JCM 12896(T)).

Bacterial Typing Techniques↗

PubMed2005

Actinoalloteichus spitiensis sp. nov., a novel actinobacterium isolated from a cold desert of the Indian Himalayas.

An actinobacterial strain, RMV-1378T, isolated from a cold desert of the Indian Himalayas, was subjected to polyphasic taxonomic characterization. The strain formed branching, non-fragmenting vegetative hyphae and did not produce diffusible pigments. Neither aerial mycelium nor spore formation was observed. The G+C content of the DNA was 72.0 mol%. The strain had chemotaxonomic characteristics typical of the genus Actinoalloteichus and was closely related (99.3 % 16S rRNA gene sequence similarity) to Actinoalloteichus cyanogriseus, currently the only Actinoalloteichus species with a validly published name. However, the results of DNA-DNA hybridization experiments showed 51.9 % relatedness with the type strain of A. cyanogriseus. On the basis of the above data and the physiological and biochemical distinctiveness of RMV-1378T (=MTCC 6194T=JCM 12472T=DSM 44848T), this strain should be classified as the type strain of a novel species of Actinoalloteichus, for which the name Actinoalloteichus spitiensis sp. nov. is proposed.

Actinomycetales↗

PubMed2003

Inherent protein structural flexibility at the RNA-binding interface of L30e.

The Saccharomyces cerevisiae ribosomal protein L30 autoregulates its own expression by binding to a purine-rich internal loop in its pre-mRNA and mRNA. NMR studies of L30 and its RNA complex showed that both the internal loop of the RNA as well as a region of the protein become substantially more ordered upon binding. A crystal structure of a maltose binding protein (MBP)-L30 fusion protein with two copies in the asymmetric unit has been determined. The flexible RNA-binding region in the L30 copies has two distinct conformations, one resembles the RNA bound form solved by NMR and the other is unique. Structure prediction algorithms also had difficulty accurately predicting this region, which is consistent with conformational flexibility seen in the NMR and X-ray crystallography studies. Inherent conformational flexibility may be a hallmark of regions involved in intermolecular interactions.

Amino Acid Sequence↗

PubMed2003

Combinatorial crystallization of an RNA-protein complex.

One of the most difficult steps in X-ray crystallography of a ribonucleoprotein (RNP) complex is obtaining crystals that diffract to high resolution. This paper describes a procedure for identifying the optimal lengths of the nucleic acid components that provide high-quality crystals of the RNP. Both strands of an RNA duplex were varied in a systematic manner to generate a large number of unique RNPs that were screened for crystallization behavior. As observed in the crystallization of other nucleic acids and their complexes, the exact length of the RNA chains was found to be critical in obtaining diffraction-quality crystals, even though the relative molecular weights of the protein and RNA components were approximately 50 and approximately 10 kDa, respectively. In particular, the helix-loop-helix structure in the mRNA for the Saccharomyces cerevisiae ribosomal protein L30, which functions as an autoregulatory element for L30 expression, was synthesized as two separate RNA chains of variable length (12-14 and 15-17 nucletides). Duplex formation of these RNAs formed the asymmetric, internal loop-binding site for L30. 16 such RNA duplexes, varying by +/-1 residue at the 5' or 3' end of either chain, were used to prepare 16 unique complexes with a maltose-binding protein-L30 fusion protein. The complexes were screened against 48 standard crystallization conditions in 2304 experiments, yielding 30 conditions with single crystals in the initial screen. The most promising of these is being used for structure determination.

Carrier Proteins↗

PubMed2003

The D1/D2 domain of the large-subunit rDNA of the yeast species Clavispora lusitaniae is unusually polymorphic.

Ten different versions of the D1/D2 divergent domain of the large-subunit ribosomal DNA were identified among interbreeding members of the yeast species Clavispora lusitaniae. One major polymorphism, located in a 90-bp structural motif of the D2 domain, exists in two versions that differ by 32 base substitutions. Three other polymorphisms consist of a two-base substitution, a two-base deletion, and a single-base deletion, respectively. The polymorphisms are independent of one another and of the two mating types, indicating that the strains studied belong to a single, sexually active Mendelian population. Several strains were heterogeneous for one or more of the polymorphisms, and one strain was found to be automictic and capable of producing asci on its own by isogamous conjugation or by bud-parent autogamy. These observations suggest circumspection in the use of sequence divergence as the principal criterion for delimiting yeast species.

DNA, Fungal↗

PubMed2003

Apophysomyces elegans: an emerging zygomycete in India.

Apophysomyces elegans was considered a rare but medically important zygomycete. We analyzed the clinical records of eight patients from a single center in whom zygomycosis due to A. elegans was diagnosed over a span of 25 months. We also attempted a DNA-based method for rapid identification of the fungi and looked for interstrain polymorphism using microsattelite primers. Three patients had cutaneous and subcutaneous infections, three had isolated renal involvement, one had rhino-orbital tissue infection, and the final patient had a disseminated infection involving the spleen and kidney. Underlying illnesses were found in two patients, one with diabetes mellitus and the other with chronic alcoholism. A history of traumatic implantation was available for three patients. All except two of the patients responded to surgical and/or medical therapy; the diagnosis for the two exceptions was made at the terminal stage of infection. Restriction enzyme (MboI, MspI, HinfI) digestion of the PCR-amplified internal transcribed spacer region helped with the rapid and specific identification of A. elegans. The strains could be divided into two groups according to their patterns, with clustering into one pattern obtained by using microsatellite [(GTG)(5) and (GAC)(5)] PCR fingerprinting. The study highlights the epidemiology, clinical spectrum, and diagnosis of emerging A. elegans infections.

Adult↗

PubMed2001

Glycine rich P-loop motif in deoxyuridine pyrophosphatase.

Deoxyuridine pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester bond of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for dTTP synthesis. Similar to other nucleotide binding proteins, dUTPase also consists of a sequence motif rich in glycine residues known as P-loop motif. The P-loop motif of the nucleotide binding proteins are involved in substrate binding, catalysis, recognition and regulation of activity. In dUTPase the function of the P-loop motif is not well understood. One of the main reasons for this limited information is the lack of the three-dimensional structure of a dUTPase enzyme with an ordered Gly-rich P-loop motif with a bound substrate and Mg(2+) ion. This review presents an insight into the role of Gly-rich P-loop motif in the function of dUTPase as revealed from the crystal structure. The analysis reveals the Gly-rich P-loop motif of dUTPase to be the longest in terms of its amino-acid composition as compared to other nucleotide binding proteins and exhibit a high-degree of sequence conservation among spectrum of species. The enzyme utilizes adaptive recognition to bind to the phosphate groups of the nucleotide. In particular, the alpha-beta phosphodiester bond adopts an unfavorable eclipsed conformation in the presence of the Gly-rich P-loop motif. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.

Amino Acid Motifs↗

PubMed2000

Structures of feline immunodeficiency virus dUTP pyrophosphatase and its nucleotide complexes in three crystal forms.

dUTP pyrophosphatase (dUTPase) cleaves the alpha-beta phosphodiester of dUTP to form pyrophosphate and dUMP, preventing incorporation of uracil into DNA and providing the substrate for thymine synthesis. Seven crystal structures of feline immunodeficiency virus (FIV) dUTPase in three crystal forms have been determined, including complexes with substrate (dUTP), product (dUMP) or inhibitor (dUDP) bound. The native enzyme has been refined at 1.40 A resolution in a hexagonal crystal form and at 2.3 A resolution in an orthorhombic crystal form. In the dUDP complex in a cubic crystal form refined at 2.5 A resolution, the C-terminal conserved P-loop motif is fully ordered. The analysis defines the roles of five sequence motifs in interaction with uracil, deoxyribose and the alpha-, beta- and gamma-phosphates. The enzyme utilizes adaptive recognition to bind the alpha- and beta-phosphates. In particular, the alpha-beta phosphodiester adopts an unfavorable eclipsed conformation in the presence of the P-loop. This conformation may be relevant to the mechanism of alpha-beta phosphodiester bond cleavage.

Animals↗

PubMed2000

Crystallization and preliminary crystallographic characterization of recombinant L-methionine-alpha-deamino-gamma-mercaptomethane lyase (methioninase).

L-Methionine-alpha-deamino-gamma-mercaptomethane lyase (rMETase) is involved in the alpha,gamma-elimination of methionine to alpha-ketobutyrate, methanethiol and ammonia. The reaction catalyzed by rMETase reduces the methionine concentration of methionine-dependent tumor cells, arresting their growth. Towards the goal of developing rMETase into an effective antitumor therapeutic and also to understand the catalytic mechanism of this enzyme, rMETase from Pseudomonas putida has been expressed, purified and crystallized. The crystals belong to space group P2(1)2(1)2 and diffract X-rays to at least 2.68 A resolution at 100 K using synchrotron radiation. The unit cell has parameters a = 152.8, b = 154.6, c = 80.8 A and contains four molecules in the asymmetric unit.

Carbon-Sulfur Lyases↗

PubMed1999

Alteration of the reduction potential of the [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I.

The [4Fe-4S](2+/+) cluster of Azotobacter vinelandii ferredoxin I (FdI) has an unusually low reduction potential (E(0')) relative to other structurally similar ferredoxins. Previous attempts to raise that E(0') by modification of surface charged residues were unsuccessful. In this study mutants were designed to alter the E(0') by substitution of polar residues for nonpolar residues near the cluster and by modification of backbone amides. Three FdI variants, P21G, I40N, and I40Q, were purified and characterized, and electrochemical E(0') measurements show that all had altered E(0') relative to native FdI. For P21G FdI and I40Q FdI, the E(0') increased by +42 and +53 mV, respectively validating the importance of dipole orientation in control of E(0'). Protein Dipole Langevin Dipole calculations based on models for those variants accurately predicted the direction of the change in E(0') while overestimating the magnitude. For I40N FdI, initial calculations based on the model predicted a +168 mV change in E(0') while a -33 mV change was observed. The x-ray structure of that variant, which was determined to 2.8 A, revealed a number of changes in backbone and side chain dipole orientation and in solvent accessibility, that were not predicted by the model and that were likely to influence E(0'). Subsequent Protein Dipole Langevin Dipole calculations (using the actual I40N x-ray structures) did quite accurately predict the observed change in E(0').

Azotobacter vinelandii↗

PubMed1999

Crystal structure of an 82-nucleotide RNA-DNA complex formed by the 10-23 DNA enzyme.

The structure of a large nucleic acid complex formed by the 10-23 DNA enzyme bound to an RNA substrate was determined by X-ray diffraction at 3.0 A resolution. The 82-nucleotide complex contains two strands of DNA and two strands of RNA that form five double-helical domains. The spatial arrangement of these helices is maintained by two four-way junctions that exhibit extensive base-stacking interactions and sharp turns of the phosphodiester backbone stabilized by metal ions coordinated to nucleotides at these junctions. Although it is unlikely that the structure corresponds to the catalytically active conformation of the enzyme, it represents a novel nucleic acid fold with implications for the Holliday junction structure.

Base Sequence↗

PubMed1999

Crystal structure of transhydrogenase domain III at 1.2 A resolution.

The nicotinamide nucleotide transhydrogenases (TH) of mitochondria and bacteria are membrane-intercalated proton pumps that transduce substrate binding energy and protonmotive force via protein conformational changes. In mitochondria, TH utilizes protonmotive force to promote direct hydride ion transfer from NADH to NADP, which are bound at the distinct extramembranous domains I and III, respectively. Domain II is the membrane-intercalated domain and contains the enzyme's proton channel. This paper describes the crystal structure of the NADP(H) binding domain III of bovine TH at 1.2 A resolution. The structure reveals that NADP is bound in a manner inverted from that previously observed for nucleotide binding folds. The non-classical binding mode exposes the NADP(H) nicotinamide ring for direct contact with NAD(H) in domain I, in accord with biochemical data. The surface of domain III surrounding the exposed nicotinamide is comprised of conserved residues presumed to form the interface with domain I during hydride ion transfer. Further, an adjacent region contains a number of acidic residues, forming a surface with negative electrostatic potential which may interact with extramembranous loops of domain II. Together, the distinctive surface features allow mechanistic considerations regarding the NADP(H)-promoted conformation changes that are involved in the interactions of domain III with domains I and II for hydride ion transfer and proton translocation.

Amino Acid Motifs↗

PubMed1999

The mechanism of aconitase: 1.8 A resolution crystal structure of the S642a:citrate complex.

The crystal structure of the S642A mutant of mitochondrial aconitase (mAc) with citrate bound has been determined at 1.8 A resolution and 100 K to capture this binding mode of substrates to the native enzyme. The 2.0 A resolution, 100 K crystal structure of the S642A mutant with isocitrate binding provides a control, showing that the Ser --> Ala replacement does not alter the binding of substrates in the active site. The aconitase mechanism requires that the intermediate product, cis-aconitate, flip over by 180 degrees about the C alpha-C beta double bond. Only one of these two alternative modes of binding, that of the isocitrate mode, has been previously visualized. Now, however, the structure revealing the citrate mode of binding provides direct support for the proposed enzyme mechanism.

Aconitate Hydratase↗

PubMed1998

A T14C variant of Azotobacter vinelandii ferredoxin I undergoes facile [3Fe-4S]0 to [4Fe-4S]2+ conversion in vitro but not in vivo.

[4Fe-4S]2+/+ clusters that are ligated by Cys-X-X-Cys-X-X-Cys sequence motifs share the general feature of being hard to convert to [3Fe-4S]+/0 clusters, whereas those that contain a Cys-X-X-Asp-X-X-Cys motif undergo facile and reversible cluster interconversion. Little is known about the factors that control the in vivo assembly and conversion of these clusters. In this study we have designed and constructed a 3Fe to 4Fe cluster conversion variant of Azotobacter vinelandii ferredoxin I (FdI) in which the sequence that ligates the [3Fe-4S] cluster in native FdI was altered by converting a nearby residue, Thr-14, to Cys. Spectroscopic and electrochemical characterization shows that when purified in the presence of dithionite, T14C FdI is an O2-sensitive 8Fe protein. Both the new and the indigenous clusters have reduction potentials that are significantly shifted compared with those in native FdI, strongly suggesting a significantly altered environment around the clusters. Interestingly, whole cell EPR have revealed that T14C FdI exists as a 7Fe protein in vivo. This 7Fe form of T14C FdI is extremely similar to native FdI in its spectroscopic, electrochemical, and structural features. However, unlike native FdI which does not undergo facile cluster conversion, the 7Fe form T14C FdI quickly converts to the 8Fe form with a high efficiency under reducing conditions.

Amino Acid Sequence↗

PubMed1997

Y13C Azotobacter vinelandii ferredoxin I. A designed [Fe-S] ligand motif contains a cysteine persulfide.

Ferredoxins that contain [4Fe-4S]2+/+ clusters often obtain three of their four cysteine ligands from a highly conserved CysXXCysXXCys sequence motif. Little is known about the in vivo assembly of these clusters and the role that this sequence motif plays in that process. In this study, we have used structure as a guide in attempts to direct the formation of a [4Fe-4S]2+/+ in the [3Fe-4S]+/0 location of native (7Fe) Azotobacter vinelandii ferredoxin I (AvFdI) by providing the correct three-dimensional orientation of cysteine ligands without introducing a CysXXCysXXCys motif. Tyr13 of AvFdI occupies the position of the fourth ligating cysteine in the homologous and structurally characterized 8Fe ferredoxin from Peptococcus aerogenes and a Y13C variant of AvFdI could be easily modeled as an 8Fe protein. However, characterization of purified Y13C FdI by UV-visible spectra, circular dichroism, electron paramagnetic resonance spectroscopies, and by x-ray crystallography revealed that the protein failed to use the introduced cysteine as a ligand and retained its [3Fe-4S]+/0 cluster. Further, electrochemical characterization showed that the redox potential and pH behavior of the cluster were unaffected by the substitution of Tyr by Cys. Although Y13C FdI is functional in vivo it does differ significantly from native FdI in that it is extremely unstable in the reduced state possibly due to increased solvent exposure of the [3Fe-4S]0 cluster. Surprisingly, the x-ray structure showed that the introduced cysteine was modified to become a persulfide. This modification may have occurred in vivo via the action of NifS, which is known to be expressed under the growth conditions used. It is interesting to note that neither of the two free cysteines present in FdI was modified. Thus, if NifS is involved in modifying the introduced cysteine there must be specificity to the reaction.

Amino Acid Sequence↗