RESUMO
As a step toward predictive modeling of flux through the pathway of monolignol biosynthesis in stem differentiating xylem of Populus trichocarpa, we discovered that the two 4-coumaric acid:CoA ligase (4CL) isoforms, 4CL3 and 4CL5, interact in vivo and in vitro to form a heterotetrameric protein complex. This conclusion is based on laser microdissection, coimmunoprecipitation, chemical cross-linking, bimolecular fluorescence complementation, and mass spectrometry. The tetramer is composed of three subunits of 4CL3 and one of 4CL5. 4CL5 appears to have a regulatory role. This protein-protein interaction affects the direction and rate of metabolic flux for monolignol biosynthesis in P. trichocarpa. A mathematical model was developed for the behavior of 4CL3 and 4CL5 individually and in mixtures that form the enzyme complex. The model incorporates effects of mixtures of multiple hydroxycinnamic acid substrates, competitive inhibition, uncompetitive inhibition, and self-inhibition, along with characteristic of the substrates, the enzyme isoforms, and the tetrameric complex. Kinetic analysis of different ratios of the enzyme isoforms shows both inhibition and activation components, which are explained by the mathematical model and provide insight into the regulation of metabolic flux for monolignol biosynthesis by protein complex formation.
Assuntos
Coenzima A Ligases/metabolismo , Ácidos Cumáricos/metabolismo , Lignina/biossíntese , Populus/metabolismo , Biologia de Sistemas , Coenzima A Ligases/genética , Imunoprecipitação , Espectrometria de Massas , Modelos Biológicos , Propionatos , RNA Mensageiro/genética , Especificidade por SubstratoRESUMO
4-Coumaric acid:coenzyme A ligase (4CL) is involved in monolignol biosynthesis for lignification in plant cell walls. It ligates coenzyme A (CoA) with hydroxycinnamic acids, such as 4-coumaric and caffeic acids, into hydroxycinnamoyl-CoA thioesters. The ligation ensures the activated state of the acid for reduction into monolignols. In Populus spp., it has long been thought that one monolignol-specific 4CL is involved. Here, we present evidence of two monolignol 4CLs, Ptr4CL3 and Ptr4CL5, in Populus trichocarpa. Ptr4CL3 is the ortholog of the monolignol 4CL reported for many other species. Ptr4CL5 is novel. The two Ptr4CLs exhibited distinct Michaelis-Menten kinetic properties. Inhibition kinetics demonstrated that hydroxycinnamic acid substrates are also inhibitors of 4CL and suggested that Ptr4CL5 is an allosteric enzyme. Experimentally validated flux simulation, incorporating reaction/inhibition kinetics, suggested two CoA ligation paths in vivo: one through 4-coumaric acid and the other through caffeic acid. We previously showed that a membrane protein complex mediated the 3-hydroxylation of 4-coumaric acid to caffeic acid. The demonstration here of two ligation paths requiring these acids supports this 3-hydroxylation function. Ptr4CL3 regulates both CoA ligation paths with similar efficiencies, whereas Ptr4CL5 regulates primarily the caffeic acid path. Both paths can be inhibited by caffeic acid. The Ptr4CL5-catalyzed caffeic acid metabolism, therefore, may also act to mitigate the inhibition by caffeic acid to maintain a proper ligation flux. A high level of caffeic acid was detected in stem-differentiating xylem of P. trichocarpa. Our results suggest that Ptr4CL5 and caffeic acid coordinately modulate the CoA ligation flux for monolignol biosynthesis.
Assuntos
Vias Biossintéticas , Coenzima A Ligases/metabolismo , Coenzima A/metabolismo , Simulação por Computador , Ácidos Cumáricos/metabolismo , Lignina/biossíntese , Populus/enzimologia , Regulação Alostérica/efeitos dos fármacos , Sítios de Ligação , Vias Biossintéticas/efeitos dos fármacos , Western Blotting , Ácidos Cafeicos/farmacologia , Coenzima A Ligases/antagonistas & inibidores , Ácidos Cumáricos/química , Ácidos Cumáricos/farmacologia , Cinética , Lignina/química , Fenilpropionatos/metabolismo , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Extratos Vegetais , Populus/efeitos dos fármacos , Propionatos , Proteômica , Proteínas Recombinantes de Fusão/metabolismo , Homologia de Sequência de Aminoácidos , Especificidade por Substrato/efeitos dos fármacos , Xilema/efeitos dos fármacos , Xilema/metabolismoRESUMO
Polymer-flooding produced water is more difficult to treat for reinjection compared with normal produced water because of the presence of residual hydrolyzed polyacrylamide (HPAM). A novel cathode membrane integrated electro-hybrid ozonation-coagulation (CM-E-HOC) process was proposed for the treatment of polymer-flooding produced water. This process achieved in situ self-cleaning by generated microbubbles in the cathode membrane. The CM-E-HOC process achieved a higher suspended solid (SS), turbidity and PAM removal efficiency than the CM-EC process. The SS in the CM-E-HOC effluent was ≤ 20 mg/L SS, which met the reinjection requirements of Longdong, Changqing Oilfield, China (Q/SYCQ 08,011-2019) at different current densities (3, 5 and 10 mA/cm2). The CM-E-HOC process greatly mitigated both reversible and irreversible membrane fouling. Therefore, excellent flux recovery was obtained at different in situ self-cleaning intervals during the CM-E-HOC process. Furthermore, alternating filtration achieved continuous water production during the CM-E-HOC process. On one hand, the effective removal of aromatic protein-like substances and an increase in oxygen-containing functional groups were achieved due to the enhanced oxidation ability of the CM-E-HOC process, which decreased membrane fouling. On the other hand, the CM-E-HOC process showed improved coagulation performance because of the increased oxygen-containing functional groups and polymeric Fe species. Therefore, larger flocs with higher fractal dimensions were generated, and a looser and more porous cake layer was formed on the membrane surface during the CM-E-HOC process. Consequently, the CM-E-HOC process exhibited better in situ self-cleaning performance and lower filtration resistance than the CM-EC process.
Assuntos
Ozônio , Purificação da Água , Polímeros , Membranas Artificiais , Filtração/métodos , Purificação da Água/métodos , OxigênioRESUMO
Flowering plants have syringyl and guaiacyl subunits in lignin in contrast to the guaiacyl lignin in gymnosperms. The biosynthesis of syringyl subunits is initiated by coniferaldehyde 5-hydroxylase (CAld5H). In Populus trichocarpa there are two closely related CAld5H enzymes (PtrCAld5H1 and PtrCAld5H2) associated with lignin biosynthesis during wood formation. We used yeast recombinant PtrCAld5H1 and PtrCAld5H2 proteins to carry out Michaelis-Menten and inhibition kinetics with LC-MS/MS based absolute protein quantification. CAld5H, a monooxygenase, requires a cytochrome P450 reductase (CPR) as an electron donor. We cloned and expressed three P. trichocarpa CPRs in yeast and show that all are active with both CAld5Hs. The kinetic analysis shows both CAld5Hs have essentially the same biochemical functions. When both CAld5Hs are coexpressed in the same yeast membranes, the resulting enzyme activities are additive, suggesting functional redundancy and independence of these two enzymes. Simulated reaction flux based on Michaelis-Menten kinetics and inhibition kinetics confirmed the redundancy and independence. Subcellular localization of both CAld5Hs as sGFP fusion proteins expressed in P. trichocarpa differentiating xylem protoplasts indicate that they are endoplasmic reticulum resident proteins. These results imply that during wood formation, 5-hydroxylation in monolignol biosynthesis of P. trichocarpa requires the combined metabolic flux of these two CAld5Hs to maintain adequate biosynthesis of syringyl lignin. The combination of genetic analysis, absolute protein quantitation-based enzyme kinetics, homologous CPR specificity, SNP characterization, and ER localization provides a more rigorous basis for a comprehensive systems understanding of 5-hydroxylation in lignin biosynthesis.
Assuntos
Lignina/biossíntese , Oxigenases de Função Mista/metabolismo , Populus/metabolismo , Xilema/enzimologia , Clonagem Molecular , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Hidroxilação , Cinética , Lignina/análise , Plantas Geneticamente Modificadas , Leveduras/metabolismoRESUMO
A multi-omics quantitative integrative analysis of lignin biosynthesis can advance the strategic engineering of wood for timber, pulp, and biofuels. Lignin is polymerized from three monomers (monolignols) produced by a grid-like pathway. The pathway in wood formation of Populus trichocarpa has at least 21 genes, encoding enzymes that mediate 37 reactions on 24 metabolites, leading to lignin and affecting wood properties. We perturb these 21 pathway genes and integrate transcriptomic, proteomic, fluxomic and phenomic data from 221 lines selected from ~2000 transgenics (6-month-old). The integrative analysis estimates how changing expression of pathway gene or gene combination affects protein abundance, metabolic-flux, metabolite concentrations, and 25 wood traits, including lignin, tree-growth, density, strength, and saccharification. The analysis then predicts improvements in any of these 25 traits individually or in combinations, through engineering expression of specific monolignol genes. The analysis may lead to greater understanding of other pathways for improved growth and adaptation.