Crystal structure of uncleaved L-aspartate-alpha-decarboxylase from Mycobacterium tuberculosis

L-aspartate-alpha-decarboxylase (ADC) is a critical regulatory enzyme in the pantothenate biosynthetic pathway and belongs to a small class of self-cleaving and pyruvoyl-dependent amino acid decarboxylases. The expression level of ADC in Mycobacterium tuberculosis (Mtb) was confirmed by cDNA analysis, immunoblotting with an anti-ADC polyclonal antibody using whole cell lysate and immunoelectron microscopy. The recombinant ADC proenzyme from Mycobacterium tuberculosis (MtbADC) was overexpressed in E. coli and the protein structure was determined at 2.99 A resolution. The proteins fold into the double-psi beta-barrel structure. The subunits of the two tetramers (there are eight ADC molecules in the asymmetric unit) form pseudo fourfold rotational symmetry, similar to the E. coli ADC proenzyme structure. As pantothenate is synthesized in microorganisms, plants, and fungi but not in animals, structure elucidation of Mtb ADC is of substantial interest for structure-based drug development.     

Comparative Genomics and Synteny Analysis of <Emphasis Type="BoldItalic">KCS17</Emphasis>-<Emphasis Type="BoldItalic">KCS18</Emphasis> Cluster Across Different Genomes and Sub-genomes of Brassicaceae for Analysis of Its Evolutionary History

Comparative genomics-based synteny analysis has proved to be an effective strategy to understand evolution of genomic regions spanning a single gene (micro-unit) to large segments encompassing hundreds of kilobases to megabases. Brassicaceae is in a unique position to contribute to understanding genome and trait evolution through comparative genomics because whole genome sequences from as many as nine species have been completed and are available for analysis. In the present work, we compared genomic loci surrounding the KCS17-KCS18 cluster across these nine genomes. KCS18 or FAE 1 gene encodes beta-ketoacyl synthase, (β-KCS) a membrane-bound enzyme that catalyses the key rate-limiting step during synthesis of VLCFAs such as erucic acid (C22) present in seed oil in Brassicaceae by elongating carbon chain from C18 to C22; knowledge on role of KCS17 in plant development is however lacking. Synteny across the genomic segments harbouring FAE1 showed variable levels of gene retention ranging between 26% (Arabidopsis thaliana and Brassica napus C03) and 89% (between A. thaliana and Brassica rapa A01), and gene density ranged between 1 gene/2.86 kb and 1 gene/4.88 kb. Interestingly, in diploid Brassica species, FAE1 was retained in only one of the sub-genomes in spite of the presence of three sub-genomes created as a result of genome triplication; in contrast, FAE1 was present at three loci, with four copies in Camellina sativa which is also known to have experienced a recent genome triplication revealing contrasting fates upon duplication. The organization of KCS17 and KCS18 as head-to-tail cluster was conserved across most of the species, except the C genome containing Brassicas, namely B. oleracea and B. napus, where disruptions because of other genes were observed. Even in the conserved blocks, the distance between KCS17 and KCS18 varied; the functional implication of the organization of KCS17-KCS18 as a cluster vis-à-vis fatty acid biosynthesis needs to be dissected, as the cis-regulatory region is expected to be present in the intergenic space. Phylogenetic analysis of KCS gene family along with KCS17-KCS18 from members of Brassicaceae reveals their ancestral relationship with KCS8-KCS9 block. Further comparative functional analysis between KCS8, KCS9, KCS16, KCS17 and KCS18 across evolutionary time-scale will be required to understand the conservation or diversification of roles of these members of KCS family in fatty acid biosynthesis during course of evolution.     

Synthesis, molecular modeling and bio-evaluation of cycloalkyl fused 2-aminopyrimidines as antitubercular and antidiabetic agents

An economical and efficient one step synthesis of a series of 8-(arylidene)-4-(aryl)-5,6,7,8-tetrahydro-quinazolin-2-ylamines and 9-(arylidene)-4-(aryl)-6,7,8,9-tetrahydro-5H-cycloheptapyrimidin-2-ylamines by the reaction of bis-benzylidene cycloalkanones and guanidine hydrochloride in presence of NaH has been developed. All the synthesized compounds were evaluated against Mycobacterium tuberculosis H₃₇Rv strain and the α-glucosidase and glycogen phosphorylase enzymes. Few of the compounds have shown interesting in vitro activity with MIC up to 3.12 μg/mL against M. tuberculosis and very good inhibition of α-glucosidase and glycogen phosphorylase enzymes. The most potent non toxic compound 40 exhibited about 58% ex vivo activity at MIC of 3.12 μg/mL. The present study opens a new gate to synthesize antitubercular agents for diabetic TB patients. In silico docking studies indicate that mycobacterial dihydrofolate reductase is the possible target of these compounds.     

Improved performance of preordered fungal protease-stearic acid biocomposites: enhanced catalytic activity,reusability, and temporal stability

In an earlier report on fungal protease (F-prot)-fatty acid biocomposite film formation [Gole et al. Anal. Chem. 2000, 72, 4301], it was observed that the biocatalytic activity of the immobilized enzyme was comparable to that of the free enzyme in solution. However, a somewhat negative aspect of the protocol was the steady loss in activity during reuse and storage of the biocomposite film. In this paper, we address the latter issues and demonstrate successful attempts toward the realization of efficient biocomposite films with enhanced biological activity, temporal stability, and excellent reusability. The improved performance of the F-prot-stearic acid biocomposite is accomplished by preordering the fatty acid film by incorporation of Pb(2+) ions into the lipid matrix prior to enzyme immobilization. The lead cation induces lamellar ordering in the lipid film and thus facilitates diffusion of the F-prot molecules into the lipid matrix and accessibility of the substrate molecules (hemoglobin, Hb) to the entrapped F-prot enzyme molecules. The preordering consequently leads to effective control of the "mass transport" problem and might be responsible for the enhanced biological activity ( approximately 36%) of the enzyme molecules in the biocomposite in comparison with the free enzyme in solution, as well the excellent reusability of the composite film. In addition to biocatalytic activity measurements, the formation and characterization of the F-prot-lead stearate biocomposite films was done by quartz crystal microgravimetry and X-ray diffraction.     

Active Site Coupling in PDE:PKA Complexes Promotes Resetting of Mammalian cAMP Signaling

Cyclic 3′5′ adenosine monophosphate (cAMP)-dependent-protein kinase (PKA) signaling is a fundamental regulatory pathway for mediating cellular responses to hormonal stimuli. The pathway is activated by high-affinity association of cAMP with the regulatory subunit of PKA and signal termination is achieved upon cAMP dissociation from PKA. Although steps in the activation phase are well understood, little is known on how signal termination/resetting occurs. Due to the high affinity of cAMP to PKA (K_D ∼ low nM), bound cAMP does not readily dissociate from PKA, thus begging the question of how tightly bound cAMP is released from PKA to reset its signaling state to respond to subsequent stimuli. It has been recently shown that phosphodiesterases (PDEs) can catalyze dissociation of bound cAMP and thereby play an active role in cAMP signal desensitization/termination. This is achieved through direct interactions with the regulatory subunit of PKA, thereby facilitating cAMP dissociation and hydrolysis. In this study, we have mapped direct interactions between a specific cyclic nucleotide phosphodiesterase (PDE8A) and a PKA regulatory subunit (RIα isoform) in mammalian cAMP signaling, by a combination of amide hydrogen/deuterium exchange mass spectrometry, peptide array, and computational docking. The interaction interface of the PDE8A:RIα complex, probed by peptide array and hydrogen/deuterium exchange mass spectrometry, brings together regions spanning the phosphodiesterase active site and cAMP-binding sites of RIα. Computational docking combined with amide hydrogen/deuterium exchange mass spectrometry provided a model for parallel dissociation of bound cAMP from the two tandem cAMP-binding domains of RIα. Active site coupling suggests a role for substrate channeling in the PDE-dependent dissociation and hydrolysis of cAMP bound to PKA. This is the first instance, to our knowledge, of PDEs directly interacting with a cAMP-receptor protein in a mammalian system, and highlights an entirely new class of binding partners for RIα. This study also highlights applications of structural mass spectrometry combined with computational docking for mapping dynamics in transient signaling protein complexes. Together, these results present a novel and critical role for phosphodiesterases in moderating local concentrations of cAMP in microdomains and signal resetting.     

Effects of GA3 and IBA on rooting and on the sprouting of buds on stem cuttings of Ipomoea fistulosa

Both IBA and GA₃ enhance rooting, IBA being more effective particularlyat a concentration of 100 mg/liter as compared to 10 mg/liter.IBA decreases while GA₃ increases the sprouting of buds, withboth effects increasing with concentration. GA₃-caused promotionof rooting appears to be due to an increased synthesis of auxinand to an adequate supply of mobilized food materials. ¹ Present address: Department of Biology, University of Missouri,St. Louis, U.S.A. (Received January 26, 1972; )     

A 3.5 genome equivalent multi access YAC library: construction, characterisation, screening and storage.

The construction of a yeast artificial chromosome (YAC) primary gridded library of 35,000 clones from human lymphoblastoid (48,XXXX) cell line DNA is described. The average YAC size is approximately 350kb representing a greater than 3.5 times coverage of the genome. The library is stored at -70 degrees C as gridded clones on nylon filters impregnated with 20% glycerol and as glycerol suspensions of individual clones in microtitre plates providing a prolonged multi-user potential. To date we have used 14 single copy probes to screen this library by colony hybridisation as well as PCR and have isolated between 1 and 5 YAC clones for every probe.     

A novel, rapid method for the isolation of terminal sequences from yeast artificial chromosome (YAC) clones.

The recent development of yeast artificial chromosome (YAC) vectors has provided a system for cloning fragments that are over ten times larger than those that can be cloned in more established systems. We have developed a method for the rapid isolation of terminal sequences from YAC clones. The YAC clone is digested with a range of restriction enzymes, a common linker is ligated to the DNA fragments and terminal sequences are amplified using a vector specific primer and a linker specific primer. Sequence data derived from these terminal specific products can be used to design primers for a further round of screening to isolate overlapping clones. The method also provides a convenient method of generating Sequence Tagged Sites for the mapping of complex genomes.