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Sarah J. Awan - One of the best experts on this subject based on the ideXlab platform.

Peter M Shoolinginjordan - One of the best experts on this subject based on the ideXlab platform.

  • insights into the mechanism of pyrrole polymerization catalysed by porphobilinogen deaminase high resolution x ray studies of the arabidopsis thaliana enzyme
    Acta Crystallographica Section D-biological Crystallography, 2013
    Co-Authors: A Roberts, P T Erskine, J B Cooper, S P Wood, R Gill, R J Hussey, Halina Mikolajek, E J T Chrystal, Peter M Shoolinginjordan
    Abstract:

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses a key early step of the haem- and chlorophyll-biosynthesis pathways in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The active site possesses an unusual Dipyrromethane cofactor which is extended during the reaction by the sequential addition of the four substrate molecules. The cofactor is linked covalently to the enzyme through a thioether bridge to the invariant Cys254. Until recently, structural data have only been available for the Escherichia coli and human forms of the enzyme. The expression of a codon-optimized gene for PBGD from Arabidopsis thaliana (thale cress) has permitted for the first time the X-ray analysis of the enzyme from a higher plant species at 1.45 A resolution. The A. thaliana structure differs appreciably from the E. coli and human forms of the enzyme in that the active site is shielded by an extensive well defined loop region (residues 60–70) formed by highly conserved residues. This loop is completely disordered and uncharacterized in the E. coli and human PBGD structures. The new structure establishes that the Dipyrromethane cofactor of the enzyme has become oxidized to the dipyrromethenone form, with both pyrrole groups approximately coplanar. Modelling of an intermediate of the elongation process into the active site suggests that the interactions observed between the two pyrrole rings of the cofactor and the active-site residues are highly specific and are most likely to represent the catalytically relevant binding mode. During the elongation cycle, it is thought that domain movements cause the bound cofactor and polypyrrole intermediates to move past the catalytic machinery in a stepwise manner, thus permitting the binding of additional substrate moieties and completion of the tetrapyrrole product. Such a model would allow the condensation reactions to be driven by the extensive interactions that are observed between the enzyme and the Dipyrromethane cofactor, coupled with acid–base catalysis provided by the invariant aspartate residue Asp95.

  • human porphobilinogen deaminase mutations in the investigation of the mechanism of Dipyrromethane cofactor assembly and tetrapyrrole formation
    Biochemical Society Transactions, 2003
    Co-Authors: Peter M Shoolinginjordan, A Aldbass, L A Mcneill, M Sarwar, D Butler
    Abstract:

    Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of Dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the Dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the Dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for Dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.

Martin J. Warren - One of the best experts on this subject based on the ideXlab platform.

  • structural evidence for the partially oxidized dipyrromethene and dipyrromethanone forms of the cofactor of porphobilinogen deaminase structures of the bacillus megaterium enzyme at near atomic resolution
    Acta Crystallographica Section D-biological Crystallography, 2014
    Co-Authors: N Azim, Martin J. Warren, P T Erskine, J B Cooper, Evelyne Deery, S P Wood, Alun R Coker, B A A Wolfenden, M Akhtar
    Abstract:

    The enzyme porphobilinogen deaminase (PBGD; hydroxymethylbilane synthase; EC 2.5.1.61) catalyses an early step of the tetrapyrrole-biosynthesis pathway in which four molecules of the monopyrrole porphobilinogen are condensed to form a linear tetrapyrrole. The enzyme possesses a Dipyrromethane cofactor, which is covalently linked by a thioether bridge to an invariant cysteine residue (Cys241 in the Bacillus megaterium enzyme). The cofactor is extended during the reaction by the sequential addition of the four substrate molecules, which are released as a linear tetrapyrrole product. Expression in Escherichia coli of a His-tagged form of B. megaterium PBGD has permitted the X-ray analysis of the enzyme from this species at high resolution, showing that the cofactor becomes progressively oxidized to the dipyrromethene and dipyrromethanone forms. In previously solved PBGD structures, the oxidized cofactor is in the dipyromethenone form, in which both pyrrole rings are approximately coplanar. In contrast, the oxidized cofactor in the B. megaterium enzyme appears to be in the dipyrromethanone form, in which the C atom at the bridging α-position of the outer pyrrole ring is very clearly in a tetrahedral configuration. It is suggested that the pink colour of the freshly purified protein is owing to the presence of the dipyrromethene form of the cofactor which, in the structure reported here, adopts the same conformation as the fully reduced Dipyrromethane form.

  • Dipyrromethane cofactor assembly of porphobilinogen deaminase: Formation of apoenzyme and preparation of holoenzyme
    Methods in enzymology, 1997
    Co-Authors: Peter M. Shoolingin-jordan, Martin J. Warren, Sarah J. Awan
    Abstract:

    Publisher Summary This chapter outlines the methods for generating the apodeaminase and regenerating the holoenzyme from the apoenzyme. The methods may be adapted for labeling the Dipyrromethane cofactor with either radioactive or stable isotopes. Porphobilinogen deaminase catalyzes the formation of preuroporphyrinogen from four molecules of porphobilinogen. Preuroporphyrinogen is a highly unstable 1-hydroxymethylbilane, which acts as the substrate for uroporphyrinogen III synthase to yield uroporphyrinogen III—the common tetrapyrrole precursor for other tetrapyrroles. Porphobilinogen deaminases have been isolated from a number of sources and their properties are well established. In the holoenzyme, the four carboxylic acid groups of the Dipyrromethane cofactor interact with highly conserved arginine residues in the Escherichia coli enzyme. Porphobilinogen deaminase lacking the cofactor, termed the “apoenzyme,” may be isolated from genetically engineered bacterial strains in which the ability to synthesize the early precursors, 5-aminolevulinic acid and porphobilinogen, has been disrupted. Preuroporphyrinogen is not only the product of porphobilinogen deaminase and the substrate for uroporphyrinogen III synthase but also the precursor of the Dipyrromethane cofactor.

Ravikanth M - One of the best experts on this subject based on the ideXlab platform.

  • Synthesis and studies of crowned dipyrromethenes based macrocycles
    PERGAMON-ELSEVIER SCIENCE LTD, 2019
    Co-Authors: Ojha B, Kumar A, Kg Thorat, Ravikanth M
    Abstract:

    Three different meso-substituted new type of crowned dipyrromethene macrocycles were synthesized in decent yields over a sequence of four simple reaction steps using readily available precursors. The macrocycles were thoroughly characterized by HRMS as well as 1D and 2D NMR spectroscopic techniques and the structures of one of the macrocyles in its neutral form and protonated form were determined by X-ray crystallography. The crystal analysis indicated that the macrocycle was distorted in both neutral and protonated forms owing to the presence of flexible aliphatic ether chain. Absorption and electrochemical studies indicated that the properties of macrocycles were dependent on type of aryl group present at the meso-position of dipyrrin moiety of crowned dipyrromethene macrocycle. The preliminary studies indicated that the crowned dipyrromethene macrocycles can act as specific colorimetric optical sensor for Cu2+ ion. (C) 2019 Elsevier Ltd. All rights reserved

  • Directly Connected AzaBODIPY-BODIPY Dyad: Synthesis, Crystal Structure, and Ground- and Excited-State Interactions
    'American Chemical Society (ACS)', 2015
    Co-Authors: Kumar S, Habtom B. Gobeze, Chatterjee T, D'souza F, Ravikanth M
    Abstract:

    Directly connected, strongly interacting sensitizer donor-acceptor dyads mimic light-induced photochemical events of photosynthesis. Here, we devised a dyad composed of BF2-chelated dipyrromethene (BODIPY) directly linked to BF2-chelated tetraarylazadipyrromethene (azaBODIPY) through the beta-pyrrole position of azaBODIPY. Structural integrity of the dyad was arrived from two-dimensional NMR spectral studies, while single-crystal X-ray structure of the dyad provided the relative orientation of the two macrocycles to be similar to 62 degrees. Because of direct linking of the two entities, ultrafast energy transfer from the (BODIPY)-B-1* to azaBODIPY was witnessed. A good agreement between the theoretically estimated Forster energy transfer rate and experimentally determined rate was observed, and this rate was found to be higher than that reported for BODIPY-azaBODIPY analogues connected with spacer units. In agreement with the free-energy calculations, the product of energy transfer, (1)azaBODIPY* revealed additional photochemical events such as electron transfer leading to the creation of BODIPY center dot+-azaBODIPY(center dot-) radical ion pair, more so in polar benzonitrile than in nonpolar toluene, as evidenced by femtosecond transient spectroscopic studies. Additionally, the spectral, electrochemical, and photochemical studies of the precursor compound azaBODIPY-Dipyrromethane also revealed occurrence of excited-state events. In this case, electron transfer from the (1)azaBODIPY* to Dipyrromethane (DPM) yielded DPM center dot+-azaBODIPY(center dot-) charge-separated state. The study described here stresses the role of close association of the donor and acceptor entities to promote ultrafast photochemical events, applicable of building fast-response optoelectronic and energy-harvesting devices

  • Hexa boron-dipyrromethene cyclotriphosphazenes: synthesis, crystal structure and photophysical properties
    American Chemical Society, 2010
    Co-Authors: Rao M. Rajeswara, Bolligarla R., Butcher, Ray J., Ravikanth M
    Abstract:

    We have synthesized four examples of a cyclotriphosphazene ring appended with six boron-dipyrromethene dyes N3P3(BODIPY)6 by adopting two different methods. In method I, 1 equiv of N3P3Cl6 was treated with 6 equiv of meso-(o- or m- or p-hydroxyphenyl)boron-dipyrromethene in tetrahydrofuran (THF) in the presence of cesium carbonate. This afforded N3P3 (BODIPY)6 in yields ranging from 80 to 90 %. In method II, we first prepared hexakis(p-formylphenoxy)cyclotriphosphazene N3P3(CHO)6 by treating 1 equiv of N3P3Cl6 with 6 equiv of 4-hydroxybenzaldehyde in the presence of cesium carbonate in THF. In the second step, N3P3(CHO)6 was condensed with excess of pyrrole in the presence of catalytic amount of trifluoroacetic acid (TFA) in CH2Cl2 at room temperature and afforded hexakis(p-phenoxy Dipyrromethane)cyclotriphosphazene. In the last step, the hexakis(p-phenoxy Dipyrromethane)cyclotriphosphazene was first oxidized with 6 equiv of DDQ in CH2Cl2 at room temperature for 1 h followed by neutralization with triethylamine and further reaction with excess BF3•Et2O afforded the target N3P3(BODIPY)6 in 16% yield. The route II was used only for the synthesis of one target compound whereas the route I was used for the synthesis of all four target compounds. The four compounds were characterized by mass, NMR, absorption, electrochemical, and fluorescence techniques. The crystal structure solved for one of the compounds revealed that the P3N3 ring is slightly puckered and the six substituents were not interacting with each other and attained pseudo-axial and pseudo-equatorial positions. The photophysical studies in five different solvents indicated that the compounds exhibit large Stokes’ shifts unlike reference monomeric BODIPYs indicating that the compounds are promising for fluorescence bioassays. The quantum yields and lifetimes of compounds 1−4 depends on the type of BODIPY unit attached to the cyclotriphosphazene ring

D Butler - One of the best experts on this subject based on the ideXlab platform.

  • The Behaviour of Enzymes in Cells 731
    2016
    Co-Authors: P. M. Shoolingin-jordan, L A Mcneill, M Sarwar, A. Al-dbass, D Butler
    Abstract:

    Human porphobilinogen deaminase mutations in the investigation of the mechanism of Dipyrromethane cofactor assembly and tetrapyrrole formatio

  • human porphobilinogen deaminase mutations in the investigation of the mechanism of Dipyrromethane cofactor assembly and tetrapyrrole formation
    Biochemical Society Transactions, 2003
    Co-Authors: Peter M Shoolinginjordan, A Aldbass, L A Mcneill, M Sarwar, D Butler
    Abstract:

    Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of Dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo-protein that exists as a complex with two substrate molecules covalently bound to the Dipyrromethane cofactor arising from the reaction between the apo-protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the Dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo-protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo-protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for Dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo-enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme–substrate intermediates from the catalytic cycle.