ORCID Profile
0000-0001-8522-7658
Current Organisation
University of Adelaide
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Publisher: MDPI AG
Date: 28-01-2023
DOI: 10.3390/IJMS24032519
Abstract: Platelets play a vital role in regulating hemostasis and thrombosis. Rho GTPases are well known as molecular switches that control various cellular functions via a balanced GTP-binding/GTP-hydrolysis cycle and signaling cascade through downstream effectors. In platelets, Rho GTPases function as critical regulators by mediating signal transduction that drives platelet activation and aggregation. Mostly by gene targeting and pharmacological inhibition approaches, Rho GTPase family members RhoA, Rac1, and Cdc42 have been shown to be indispensable in regulating the actin cytoskeleton dynamics in platelets, affecting platelet shape change, spreading, secretion, and aggregation, leading to thrombus formation. Additionally, studies of Rho GTPase function using platelets as a non-transformed model due to their anucleated nature have revealed valuable information on cell signaling principles. This review provides an updated summary of recent advances in Rho GTPase signaling in platelet regulation. We also highlight pharmacological approaches that effectively inhibited platelet activation to explore their possible development into future antiplatelet therapies.
Publisher: Wiley
Date: 11-11-2022
Abstract: The cytochrome P450 family of monooxygenase enzymes have essential biological roles involving the selective oxidation of carbon‐hydrogen bonds. They can also catalyze other important metabolic reactions including desaturation to form alkenes. Currently the factors that control the partition between P450 hydroxylation and desaturation pathways are poorly defined. The CYP199A4 enzyme from the bacterium Rhodopseudomonas palustris HaA2 catalyzes the oxidation of 4‐ethyl‐ and 4‐isopropyl‐ benzoic acids with hydroxylation and desaturation occurring in significant quantities. Here we demonstrate that 4‐cyclopropylbenzoic acid is regioselectively hydroxylated by CYP199A4 at the benzylic carbon. In contrast, the oxidation of 4‐ n ‐propylbenzoic acid by CYP199A4 results in three major metabolites: an alkene from desaturation and two hydroxylation products at the benzylic (Cα) and Cβ carbons in similar quantities. Extending the length of the alkyl substituent resulted in 4‐ n ‐butylbenzoic acid being oxidized at the benzylic position (45%) and desaturated (55%). In contrast, 4‐isobutylbenzoic generated very little alkene (5%) but was hydroxylated at the benzylic position (54%) and at the tertiary Cβ position (41%). The oxidation of 4‐ n ‐propylbenzoic acid by the F298 V mutant of CYP199A4 occurred with no hydroxylation at Cβ and a significant increase in metabolites arising from desaturation (73%). The X‐ray crystal structures of CYP199A4 with each substrate revealed that they bind in the active site with the alkyl substituent positioned over the heme. However, the longer alkylbenzoic acids were bound in a different conformation as was 4‐ n ‐propylbenzoic acid in the F298 V mutant. Overall, the changes in metabolite distribution could be ascribed to bond strength differences and the position of the alkyl group relative to the heme.
Publisher: Wiley
Date: 06-10-2022
Abstract: The cytochrome P450 (CYP) family of heme monooxygenases catalyse the selective oxidation of C-H bonds under ambient conditions. The CYP199A4 enzyme from Rhodopseudomonas palustris catalyses aliphatic oxidation of 4-cyclohexylbenzoic acid but not the aromatic oxidation of 4-phenylbenzoic acid, due to the distinct mechanisms of aliphatic and aromatic oxidation. The aromatic substrates 4-benzyl-, 4-phenoxy- and 4-benzoyl-benzoic acid and methoxy-substituted phenylbenzoic acids were assessed to see if they could achieve an orientation more amenable to aromatic oxidation. CYP199A4 could catalyse the efficient benzylic oxidation of 4-benzylbenzoic acid. The methoxy-substituted phenylbenzoic acids were oxidatively demethylated with low activity. However, no aromatic oxidation was observed with any of these substrates. Crystal structures of CYP199A4 with 4-(3'-methoxyphenyl)benzoic acid demonstrated that the substrate binding mode was like that of 4-phenylbenzoic acid. 4-Phenoxy- and 4-benzoyl-benzoic acid bound with the ether or ketone oxygen atom hydrogen-bonded to the heme aqua ligand. We also investigated whether the substitution of phenylalanine residues in the active site could permit aromatic hydroxylation. Mutagenesis of the F298 residue to a valine did not significantly alter the substrate binding position or enable the aromatic oxidation of 4-phenylbenzoic acid however the F182L mutant was able to catalyse 4-phenylbenzoic acid oxidation generating 2'-hydroxy-, 3'-hydroxy- and 4'-hydroxy metabolites in a 83 : 9 : 8 ratio, respectively. Molecular dynamics simulations, in which the distance and angle of attack were considered, demonstrated that in the F182L variant, in contrast to the wild-type enzyme, the phenyl ring of 4-phenylbenzoic acid attained a productive geometry for aromatic oxidation to occur.
Publisher: Royal Society of Chemistry (RSC)
Date: 2023
DOI: 10.1039/D3CC02312E
Abstract: Cholesterol catabolism is an important survival mechanism for the pathogenic Mycobacterium tuberculosis . Here we demonstrate that the CYP125 family of cytochrome P450 enzymes can catalyse the oxidation of phytosterols as well as cholesterol.
Publisher: American Chemical Society (ACS)
Date: 26-07-2022
DOI: 10.1021/ACSINFECDIS.2C00215
Abstract: The steroid binding CYP142 cytochrome P450 enzymes of
Publisher: American Chemical Society (ACS)
Date: 06-01-2022
No related grants have been discovered for Daniel Doherty.