Nitrile biotransformationby aspergillus niger

A nitrile-converting enzyme activity was induced in Aspergillus niger K10 by 3-cyanopyridine. The whole cell biocatalyst was active at pH 3–11 and hydrolyzed the cyano group into acid and/or amide functions in benzonitrile as well as in its meta- and para-substituted derivatives, cyanopyridines, 2-phenylacetonitrile and thiophen-2-acetonitrile. Amides constituted a significant part of the total biotransformation products of 2- and 4-cyanopyridine, 4-chlorobenzonitrile, 4-tolunitrile and 1,4-dicyanobenzene, while α-substituted acrylonitriles gave amides as the sole product

Biotransformation of nitriles to amides using soluble and immobilized nitrile hydratase from Rhodococcus erythropolis A4

A semi-purified nitrile hydratase from Rhodococcus erythropolis A4 was applied to biotransformations of 3-oxonitriles 1a–4a, 3-hydroxy-2-methylenenitriles 5a–7a, 4-hydroxy-2-methylenenitriles 8a–9a, 3-hydroxynitriles 10a–12a and 3-acyloxynitrile 13a into amides 1b–13b. Cross-linked enzyme aggregates (CLEAs) with nitrile hydratase and amidase activities (88% and 77% of the initial activities, respectively) were prepared from cell-free extract of this microorganism and used for nitrile hydration in presence of ammonium sulfate, which selectively inhibited amidase activity. The genes nha1 and nha2 coding for α and β subunits of nitrile hydratase were cloned and sequenced

Kinetic Analysis of a Globin-Coupled Histidine Kinase, AfGcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

International audienceThe globin-coupled histidine kinase, AfGcHK, is a part of the two-component signal transduction system from the soil bacterium Anaeromyxobacter sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated AfGcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The kcat values of the heme Fe(III)-OH–, Fe(III)-cyanide, Fe(III)-imidazole, and Fe(II)-O2 bound active AfGcHK forms were 1.1–1.2 min–1, and their KmATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe(II) heme had a kcat of 1.0 min–1 but a very high KmATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe(II) heme-bound inactive form had kcat and KmATP values of 0.4 min–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe(III) form. The heme-free form exhibited low activity, with kcat and KmATP values of 0.3 min–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its kcat/KmATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discusse

Human Stress-inducible Hsp70 Has a High Propensity to Form ATP-dependent Antiparallel Dimers That Are Differentially Regulated by Cochaperone Binding

International audienc

Kinetic Analysis of a Globin-Coupled Histidine Kinase, <i>Af</i>GcHK: Effects of the Heme Iron Complex, Response Regulator, and Metal Cations on Autophosphorylation Activity

The globin-coupled histidine kinase, <i>Af</i>GcHK, is a part of the two-component signal transduction system from the soil bacterium <i>Anaeromyxobacter</i> sp. Fw109-5. Activation of its sensor domain significantly increases its autophosphorylation activity, which targets the His183 residue of its functional domain. The phosphate group of phosphorylated <i>Af</i>GcHK is then transferred to the cognate response regulator. We investigated the effects of selected variables on the autophosphorylation reaction’s kinetics. The <i>k</i>_cat values of the heme Fe­(III)-OH^–, Fe­(III)-cyanide, Fe­(III)-imidazole, and Fe­(II)-O₂ bound active <i>Af</i>GcHK forms were 1.1–1.2 min^–1, and their <i>K</i>_m^ATP values were 18.9–35.4 μM. However, the active form bearing a CO-bound Fe­(II) heme had a <i>k</i>_cat of 1.0 min^–1 but a very high <i>K</i>_m^ATP value of 357 μM, suggesting that its active site structure differs strongly from the other active forms. The Fe­(II) heme-bound inactive form had <i>k</i>_cat and <i>K</i>_m^ATP values of 0.4 min^–1 and 78 μM, respectively, suggesting that its low activity reflects a low affinity for ATP relative to that of the Fe­(III) form. The heme-free form exhibited low activity, with <i>k</i>_cat and <i>K</i>_m^ATP values of 0.3 min^–1 and 33.6 μM, respectively, suggesting that the heme iron complex is essential for high catalytic activity. Overall, our results indicate that the coordination and oxidation state of the sensor domain heme iron profoundly affect the enzyme’s catalytic activity because they modulate its ATP binding affinity and thus change its <i>k</i>_cat/<i>K</i>_m^ATP value. The effects of the response regulator and different divalent metal cations on the autophosphorylation reaction are also discussed