Spectral and kinetic characterization of 7,8-diaminopelargonic acid synthase from Mycobacterium tuberculosis

The indispensability of biotin for crucial processes like lipid biosynthesis coupled to the absence of the biotin biosynthesis pathway in humans make the enzymes of this pathway, attractive targets for development of novel drugs against numerous pathogens including M. tuberculosis. We report the spectral and kinetic characterization of the Mycobacterium tuberculosis 7,8-Diaminopelargonic acid (DAPA) synthase, the second enzyme of the biotin biosynthesis pathway. In contrast to the E. coli enzyme, no quinonoid intermediate was detected during the steady state reaction between the enzyme and S-adenosyl-L-methionine (SAM). The second order rate constant for this half of the reaction was determined to be 1.75 +/- 0.11 M-1s-1. The Km values for 7-keto-8-aminopelargonic acid (KAPA) and SAM are 2.83 microM and 308.28 microM, respectively whereas the Vmax and kcat values for the enzyme are 0.02074 micromoles/min/ml and 0.003 s-1, respectively. Our initial studies pave the way for further detailed mechanistic and kinetic characterization of the enzyme.     

Keratitis Due to the Wood Saprobic Ascomycete, Auerswaldia lignicola (Family Botryosphaeriaceae), in a Carpenter in India

Keratitis due to Auerswaldia lignicola in a 32-year-old Indian male carpenter is described. At presentation, the patient reported persistent pain and tearing (left eye) in spite of topical antimicrobial therapy for more than 3 weeks. Clinically, mycotic keratitis was suspected, and direct microscopy of corneal scrapings stained by lactophenol cotton blue and Gram stains revealed broad septate hyphae. Intensive topical antifungal therapy was then given for 15 days. The keratitis continued to progress, necessitating therapeutic penetrating keratoplasty. Following the keratoplasty, there was rapid reduction in inflammation and gradual quietening of the eye. Brown–black fungal colonies resembling Lasiodiplodia theobromae were isolated from corneal scrape and corneal button (post-surgery) material on Sabouraud glucose–neopeptone agar; however, sporulation did not occur, so the morphological identification could not be confirmed. Sequence analysis of the 18S rRNA region of extracted fungal genomic DNA yielded an identification of A. lignicola Ariyawansa, J.K. Liu & K.D. Hyde; the sequence data have been deposited in GenBank (A. lignicola strain DK/V4, accession number KC866317.1). Medical management of keratitis due to such rarely reported fungal species may be difficult, necessitating surgical procedures.     

Life table and efficacy of Mallada desjardinsi (Chrysopidae: Neuroptera), an important predator of tea red spider mite, Oligonychus coffeae (Acari: Tetranychidae)

Green lacewing, Mallada desjardinsi Navas, is an important predator of red spider mite, Oligonychus coffeae infesting tea. Life history, life table and efficacy of M. desjardinsi were determined using red spider mite as prey under laboratory conditions. Duration of development of M. desjardinsi recorded was 5.1, 13.8 and 13 days for eggs, larvae and pupae respectively, with an average of 31.9 days from egg to adult emergence. After a mean pre oviposition period of 7.1 days, a single female laid an average of 252.6 eggs in its life time. Adult longevity of the male was recorded as 39.6 days while the females lived longer (58.2 days). The life table of M. desjardinsi was characterized by an intrinsic rate of increase (r) of 0.096 day, net reproductive rate (R ₀ ) of 153.19 eggs/female, gross reproduction rate (∑mx) of 167.28 eggs/female, generation time (T) of 52.47 days, doubling time of 7.22 days and finite rate of increase(λ) of 1.1 day. The optimum predator–prey ratios were 1:50 and 1:33 under laboratory conditions however, 1:33 and 1:25 ratios were effective in green house conditions. The results of the study can be considered as a first step towards the utilization of this predator in an IPM program for the management of red spider mite infesting tea.     

Molecular characterization of Theileria orientalis causing fatal infection in crossbred adult bovines of South India

The disease condition attributed to have been caused by Theileria orientalis is generally benign. However, it is also thought that the parasite, at least some strains of it, can cause fatal disease. The present communication deals with the clinical signs, postmortem lesions and diagnosis of a fatal disease due to T. orientalis which caused mortality in crossbred adult bovines of South India. High body temperature, lacrimation, nasal discharge, swollen lymph nodes and haemoglobinuria were the symptoms observed. The postmortem lesions observed were punched out ulcers in abomasum, enlargement of spleen, massive pulmonary oedema, frothy exudates in trachea, epicardial and endocardial haemorrhage and haemorrhagic duodenitis. Peripheral blood smear examination revealed rod shaped Theileria sp. organisms. Polymerase chain reaction that amplify the T. orientalis specific P(32/33) gene, followed by cloning and sequencing, revealed maximum homology with Narathiwat (Thailand) and Jingole -1 (Indonesia) isolates which were positioned as isolate type 7 of T. orientalis.     

Investigations into Biochemical Mode of Inhibition of Guanase by Azepinomycin: Synthesis and Biochemical Screening of Several Analogues of Azepinomycin

Abstract

In an effort to explore the biochemical mode of guanase inhibition as well as the structure-activity relationships of azepinomycin, five analogues (I-V) of azepinomycin were synthesized and screened against guanase from rabbit liver. Our results suggest that while the 6-hydroxy group of azepinomycin is crucial for activity, its putative transition state mode of inhibition of guanase is questionable. The additional H-bonding sites at position 5, and hydrophobic groups in and around position 3 of azepinomycin appear to be tolerated, and may in fact enhance the potency of inhibition.     

Ligand specificity of group I biotin protein ligase of Mycobacterium tuberculosis

Background

Fatty acids are indispensable constituents of mycolic acids that impart toughness & permeability barrier to the cell envelope of M. tuberculosis. Biotin is an essential co-factor for acetyl-CoA carboxylase (ACC) the enzyme involved in the synthesis of malonyl-CoA, a committed precursor, needed for fatty acid synthesis. Biotin carboxyl carrier protein (BCCP) provides the co-factor for catalytic activity of ACC.

Methodology/Principal Findings

BPL/BirA (Biotin Protein Ligase), and its substrate, biotin carboxyl carrier protein (BCCP) of Mycobacterium tuberculosis (Mt) were cloned and expressed in E. coli BL21. In contrast to EcBirA and PhBPL, the ∼29.5 kDa MtBPL exists as a monomer in native, biotin and bio-5′AMP liganded forms. This was confirmed by molecular weight profiling by gel filtration on Superdex S-200 and Dynamic Light Scattering (DLS). Computational docking of biotin and bio-5′AMP to MtBPL show that adenylation alters the contact residues for biotin. MtBPL forms 11 H-bonds with biotin, relative to 35 with bio-5′AMP. Docking simulations also suggest that bio-5′AMP hydrogen bonds to the conserved ‘GRGRRG’ sequence but not biotin. The enzyme catalyzed transfer of biotin to BCCP was confirmed by incorporation of radioactive biotin and by Avidin blot. The K_m for BCCP was ∼5.2 µM and ∼420 nM for biotin. MtBPL has low affinity (K_b = 1.06×10⁻⁶ M) for biotin relative to EcBirA but their K_m are almost comparable suggesting that while the major function of MtBPL is biotinylation of BCCP, tight binding of biotin/bio-5′AMP by EcBirA is channeled for its repressor activity.

Conclusions/Significance

These studies thus open up avenues for understanding the unique features of MtBPL and the role it plays in biotin utilization in M. tuberculosis.