title: Understanding enzymatic mechanism and allostery using macromolecular rate theory

authors: Grout, Emily Katherine

subjects: Biochemistry

abstract: Intricate systems of regulation at many levels act to control enzyme rates to tune metabolism across biosynthetic pathways. Allostery is a ubiquitous regulatory mechanism for feedback and regulation of enzymes in biosynthetic pathways. ATP-phosphoribosyltransferase from Mycobacterium tuberculosis (mtuATP-PRT) catalyses the first committed step in de novo histidine biosynthesis, and allosteric product inhibition of ATP-PRT by L-His is key to regulating metabolic flux in this pathway. mtuATP-PRT also exhibits allosteric activation by a histidine analogue 3 (2-Thienyl)-L-alanine (TIH). This compound binds in the same allosteric site as L-His and appears to elicit a structurally identical “tensed” conformation, apparently contradicting previous suggestions that the tensed conformation drives inhibitory regulation in mtuATP-PRT. 

Through mutations adjacent to the allosteric binding site, we have interrupted allosteric inhibition by L-His while retaining allosteric activation by TIH. Kinetic assays of wild type ATP-PRT and mutants across a large temperature range with various allosteric effects, has identified dynamic changes between allosterically modulated states of the enzyme, and suggests an enthalpy-entropy (∆H‡-∆S‡) trade off occurring in allosteric states. 

Complementing our kinetic data, crystallographic analysis and molecular dynamics (MD) simulations of wild type and mutant ATP-PRT have identified two residues that differ upon the binding of TIH (in comparison to L-His binding). Glu-18 and Arg-27 rotate between ligand bound states and form interacting bonds in L-His bound structures that may serve to stabilise and reduce the overall flexibility of the hexameric conformation. This Glu-18 and Arg-27 interaction varies between L-His-bound, APO-mtuATP-PRT and TIH-bound structures, suggesting this interaction may demarcate allosteric activation and inhibition.

An evolutionary analysis of ATP-PRT was undertaken and has identified the long form of ATP-PRT as the ancestral form, and the short form ATP-PRT has subsequently co-opted a histidyl-tRNA synthetase (HisRS) gene product (HisZ) as its regulatory domain. Aminoacyl-tRNA synthetases are known to be prone to horizontal gene transfer, therefore the co-option of HisRS and the loss of the C-terminal domain in ATP-PRTs provides an evolutionary trajectory for diverse ATP-PRT isoforms.

text: Understanding enzymatic mechanism and allostery using macromolecular rate theory Intricate systems of regulation at many levels act to control enzyme rates to tune metabolism across biosynthetic pathways. Allostery is a ubiquitous regulatory mechanism for feedback and regulation of enzymes in biosynthetic pathways. ATP-phosphoribosyltransferase from Mycobacterium tuberculosis (mtuATP-PRT) catalyses the first committed step in de novo histidine biosynthesis, and allosteric product inhibition of ATP-PRT by L-His is key to regulating metabolic flux in this pathway. mtuATP-PRT also exhibits allosteric activation by a histidine analogue 3 (2-Thienyl)-L-alanine (TIH). This compound binds in the same allosteric site as L-His and appears to elicit a structurally identical “tensed” conformation, apparently contradicting previous suggestions that the tensed conformation drives inhibitory regulation in mtuATP-PRT. 

Through mutations adjacent to the allosteric binding site, we have interrupted allosteric inhibition by L-His while retaining allosteric activation by TIH. Kinetic assays of wild type ATP-PRT and mutants across a large temperature range with various allosteric effects, has identified dynamic changes between allosterically modulated states of the enzyme, and suggests an enthalpy-entropy (∆H‡-∆S‡) trade off occurring in allosteric states. 

Complementing our kinetic data, crystallographic analysis and molecular dynamics (MD) simulations of wild type and mutant ATP-PRT have identified two residues that differ upon the binding of TIH (in comparison to L-His binding). Glu-18 and Arg-27 rotate between ligand bound states and form interacting bonds in L-His bound structures that may serve to stabilise and reduce the overall flexibility of the hexameric conformation. This Glu-18 and Arg-27 interaction varies between L-His-bound, APO-mtuATP-PRT and TIH-bound structures, suggesting this interaction may demarcate allosteric activation and inhibition.

An evolutionary analysis of ATP-PRT was undertaken and has identified the long form of ATP-PRT as the ancestral form, and the short form ATP-PRT has subsequently co-opted a histidyl-tRNA synthetase (HisRS) gene product (HisZ) as its regulatory domain. Aminoacyl-tRNA synthetases are known to be prone to horizontal gene transfer, therefore the co-option of HisRS and the loss of the C-terminal domain in ATP-PRTs provides an evolutionary trajectory for diverse ATP-PRT isoforms.

year: 2022

title: An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga.

authors: Simpson, H.D.

subjects: biology

abstract: Endo-1,4-beta-xylanase (EC 3.2.1.8) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic anaerobic eubacterium which grows optimally at 80 degrees C. Activity was purified 165-fold by anion-exchange and hydroxyapatite chromatography. The enzyme has an Mr of 31,000 as determined by SDS/PAGE and 35,000 by analytical gel filtration. The optima for activity and stability for purified xylanase were between pH 5.0 and 5.5. At pH 5.5, which is the optimum pH for thermostability, t1/2 (95 degrees C) is 90 min. The thermostability was improved by immobilization of the xylanase on to porous glass beads; t1/2 (105 degrees C) is 10 min. Several additives, such as sorbitol and xylan, were also found to increase the thermostability. At 130 degrees C, the half-life of immobilized xylanase in the presence of 90% sorbitol was 1.3 min. At 130 degrees C in molten sorbitol half of the enzyme denatured rapidly, but the remainder appeared to have a half-life of about 60 min.

text: An extremely thermostable xylanase from the thermophilic eubacterium Thermotoga. Endo-1,4-beta-xylanase (EC 3.2.1.8) was isolated from the culture supernatant of Thermotoga sp. strain FjSS3-B.1, an extremely thermophilic anaerobic eubacterium which grows optimally at 80 degrees C. Activity was purified 165-fold by anion-exchange and hydroxyapatite chromatography. The enzyme has an Mr of 31,000 as determined by SDS/PAGE and 35,000 by analytical gel filtration. The optima for activity and stability for purified xylanase were between pH 5.0 and 5.5. At pH 5.5, which is the optimum pH for thermostability, t1/2 (95 degrees C) is 90 min. The thermostability was improved by immobilization of the xylanase on to porous glass beads; t1/2 (105 degrees C) is 10 min. Several additives, such as sorbitol and xylan, were also found to increase the thermostability. At 130 degrees C, the half-life of immobilized xylanase in the presence of 90% sorbitol was 1.3 min. At 130 degrees C in molten sorbitol half of the enzyme denatured rapidly, but the remainder appeared to have a half-life of about 60 min.

year: 1991

title: The state of the Rotorua Lakes in 2017

authors: Hamilton, David P.

abstract: I have been asked to give an overview of the Rotorua Te Arawa Lakes and then focus on just four of the lakes. Chris McBride will pick up other lakes.  I would like to acknowledge Warwick Silvester who set up the Lakes Chair, Simon Stuart who is working outside the rohe, on Lake Taupō, but has a great body of knowledge on the food chain in that lake, with potential applications to the Te Arawa lakes. Also to Kohji Muraoka who helped me over the last 24 hours, putting data together which enabled me to synthesise my thoughts on these lakes.

text: The state of the Rotorua Lakes in 2017 I have been asked to give an overview of the Rotorua Te Arawa Lakes and then focus on just four of the lakes. Chris McBride will pick up other lakes.  I would like to acknowledge Warwick Silvester who set up the Lakes Chair, Simon Stuart who is working outside the rohe, on Lake Taupō, but has a great body of knowledge on the food chain in that lake, with potential applications to the Te Arawa lakes. Also to Kohji Muraoka who helped me over the last 24 hours, putting data together which enabled me to synthesise my thoughts on these lakes.

year: 2017