RESUMO
Rhodococcus jostii RHA1 is able to degrade toxic compounds and accumulate high amounts of triacylglycerols (TAG) upon nitrogen starvation. These NADPH-dependent processes are essential for the adaptation of rhodococci to fluctuating environmental conditions. In this study, we used an MS-based, label-free and quantitative proteomic approach to better understand the integral response of R. jostii RHA1 to the presence of methyl viologen (MV) in relation to the synthesis and accumulation of TAG. The addition of MV promoted a decrease of TAG accumulation in comparison to cells cultivated under nitrogen-limiting conditions in the absence of this pro-oxidant. Proteomic analyses revealed that the abundance of key proteins of fatty acid biosynthesis, the Kennedy pathway, glyceroneogenesis and methylmalonyl-CoA pathway, among others, decreased in the presence of MV. In contrast, some proteins involved in lipolysis and ß-oxidation of fatty acids were upregulated. Some metabolic pathways linked to the synthesis of NADPH remained activated during oxidative stress as well as under nitrogen starvation conditions. Additionally, exposure to MV resulted in the activation of complete antioxidant machinery comprising superoxide dismutases, catalases, mycothiol biosynthesis, mycothione reductase and alkyl hydroperoxide reductases, among others. Our study suggests that oxidative stress response affects TAG accumulation under nitrogen-limiting conditions through programmed molecular mechanisms when both stresses occur simultaneously.
Assuntos
Nitrogênio/deficiência , Estresse Oxidativo/fisiologia , Paraquat/metabolismo , Rhodococcus/metabolismo , Triglicerídeos/biossíntese , Acil Coenzima A/metabolismo , Adaptação Fisiológica , Catalase/metabolismo , Cisteína/biossíntese , Ácidos Graxos/biossíntese , Glicopeptídeos/biossíntese , Inositol/biossíntese , NADP/metabolismo , Nitrogênio/metabolismo , Oxirredução/efeitos dos fármacos , Estresse Oxidativo/efeitos dos fármacos , Oxirredutases/biossíntese , Peroxirredoxinas/biossíntese , Proteoma , Rhodococcus/crescimento & desenvolvimento , Superóxido Dismutase/metabolismoRESUMO
Understanding enzymatic reactions with atomic resolution has proven in recent years to be of tremendous interest for biochemical research, and thus, the use of QM/MM methods for the study of reaction mechanisms is experiencing a continuous growth. Glycosyltransferases (GTs) catalyze the formation of glycosidic bonds, and are important for many biotechnological purposes, including drug targeting. Their reaction product may result with only one of the two possible stereochemical outcomes for the reacting anomeric center, and therefore, they are classified as either inverting or retaining GTs. While the inverting GT reaction mechanism has been widely studied, the retaining GT mechanism has always been controversial and several questions remain open to this day. In this work, we take advantage of our recent GPU implementation of a pure QM(DFT-PBE)/MM approach to explore the reaction and inhibition mechanism of MshA, a key retaining GT responsible for the first step of mycothiol biosynthesis, a low weight thiol compound found in pathogens like Mycobacterium tuberculosis that is essential for its survival under oxidative stress conditions. Our results show that the reaction proceeds via a front-side SNi-like concerted reaction mechanism (DNAN in IUPAC nomenclature) and has a 17.5 kcal/mol free energy barrier, which is in remarkable agreement with experimental data. Detailed analysis shows that the key reaction step is the diphosphate leaving group dissociation, leading to an oxocarbenium-ion-like transition state. In contrast, fluorinated substrate analogues increase the reaction barrier significantly, rendering the enzyme effectively inactive. Detailed analysis of the electronic structure along the reaction suggests that this particular inhibition mechanism is associated with fluorine's high electronegative nature, which hinders phosphate release and proper stabilization of the transition state.
Assuntos
Amidoidrolases/antagonistas & inibidores , Amidoidrolases/metabolismo , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/metabolismo , Cisteína/biossíntese , Glicopeptídeos/biossíntese , Glicosiltransferases/metabolismo , Inositol/biossíntese , Metais/metabolismo , Teoria Quântica , Biocatálise , Cisteína/química , Glicopeptídeos/química , Inositol/química , Mycobacterium tuberculosis/metabolismoRESUMO
Using as a model Mycobacterium smegmatis, a non-motile microorganism, we have studied for the first time in mycobacteria the phenomenon known as sliding motility, as well as the process of biofilm formation. A screen of random transposon mutants was performed in order to identify the genes required for mycobacterial sliding over the surface of motility plates. The genetic analysis described here has been published recently. The genes required for sliding and for biofilm formation (mps and tmtpC) are involved in the biosynthesis of the glycopeptidolipids (GPLs) and their transport to the mycobacterial capsule. Based on our results, we suggest a model for the role of the GPLs in both phenomena.