Characterization of acyl-ACP thioesterases for the purpose of diversifying fatty acid synthesis pathway

Acyl-ACP TE selectively hydrolyzes the thiol ester bonds of acyl-ACPs to release free fatty acids, and therefore plays an essential role in determining the output of fatty acid synthesis (FAS) pathway. Comprehensive understanding of acyl-ACP TE is demanded to tailor this biocatalyst for the application in metabolic engineering of FAS pathway. To explore the diversity of acyl-ACP TEs, a total of 31 TEs enzymes were sourced from a wide range of biological taxa, including plants and bacteria, and these were functionally characterized. The results demonstrate that acyl-ACP TEs have great functional diversity relative to the acyl chain length specificity as well as acyl chains that contain additional chemical functionalities. Multiple sequence alignment of plant and bacterial TEs, and structure modeling of CvFatB2 revealed that a previously proposed residue Cys348 is unlikely to be a catalytic residue. Instead, residues Asp309 and Glu347, in addition to previously proposed residues Asn311 and His313 (numbers are based on CvFatB2 sequence), were proposed to be involved in the catalysis of acyl-ACP TEs. In vivo activities of site-directed mutants proved this hypothesis, and a two-step catalytic mechanism for plant and bacterial acyl-ACP TEs is proposed. To identify the region(s) that determine the substrate specificity, two acyl-ACP TEs were used for a domain-shuffling study. Comparing the substrate specificities of the resulting chimeric TEs led to the identification of the most important region that determines the substrate specificity of acyl-ACP TE. Site-directed mutagenesis analysis proved that six residues play critical roles in determining the substrate specificity, including V194 in Fragment II, V217, N223, R226, and R227 in Fragment III, and I268 in Fragment IV. Another three residues, L257, I260, and L289, impact the catalytic activity of acyl-ACP TE, because they are in two proposed ACP binding motifs. A directed evolution approach was successfully developed to improve the fatty acid productivity of acyl-ACP TE. Screening a designed variant library resulted in recovery of TE variants with increased fatty acid productivity and more insight into the relationship between sequences and substrate specificities of acyl-ACP TE

Identification of Active Site Residues Implies a Two-step Catalytic Mechanism for Acyl-ACP Thioesterase

In plants and bacteria that use a Type II fatty acid synthase (FAS), isozymes of acyl-acyl carrier protein (ACP) thioesterase (TEs) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In this study, sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2 suggest that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hot-dog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate

Phylogenetic and experimental characterization of an acyl-ACP thioesterase family reveals significant diversity in enzymatic specificity and activity

<p>Abstract</p> <p>Background</p> <p>Acyl-acyl carrier protein thioesterases (acyl-ACP TEs) catalyze the hydrolysis of the thioester bond that links the acyl chain to the sulfhydryl group of the phosphopantetheine prosthetic group of ACP. This reaction terminates acyl chain elongation of fatty acid biosynthesis, and in plant seeds it is the biochemical determinant of the fatty acid compositions of storage lipids.</p> <p>Results</p> <p>To explore acyl-ACP TE diversity and to identify novel acyl ACP-TEs, 31 acyl-ACP TEs from wide-ranging phylogenetic sources were characterized to ascertain their <it>in vivo </it>activities and substrate specificities. These acyl-ACP TEs were chosen by two different approaches: 1) 24 TEs were selected from public databases on the basis of phylogenetic analysis and fatty acid profile knowledge of their source organisms; and 2) seven TEs were molecularly cloned from oil palm (<it>Elaeis guineensis</it>), coconut (<it>Cocos nucifera</it>) and <it>Cuphea viscosissima</it>, organisms that produce medium-chain and short-chain fatty acids in their seeds. The <it>in vivo </it>substrate specificities of the acyl-ACP TEs were determined in <it>E. coli</it>. Based on their specificities, these enzymes were clustered into three classes: 1) Class I acyl-ACP TEs act primarily on 14- and 16-carbon acyl-ACP substrates; 2) Class II acyl-ACP TEs have broad substrate specificities, with major activities toward 8- and 14-carbon acyl-ACP substrates; and 3) Class III acyl-ACP TEs act predominantly on 8-carbon acyl-ACPs. Several novel acyl-ACP TEs act on short-chain and unsaturated acyl-ACP or 3-ketoacyl-ACP substrates, indicating the diversity of enzymatic specificity in this enzyme family.</p> <p>Conclusion</p> <p>These acyl-ACP TEs can potentially be used to diversify the fatty acid biosynthesis pathway to produce novel fatty acids.</p

Comparative transcriptomic analysis reveals the molecular mechanisms related to oxytetracycline- resistance in strains of Aeromonas hydrophila

Antibiotic resistance among aquatic bacterial pathogens has become a serious concern in aquaculture environments, which has increased research interest to develop solutions to overcome this problem. Moreover, the activities of several Aeromonas hydrophila gene pathways have remained elusive concerning antibiotic resistance evolution. Therefore, in this study, we have performed a transcriptomic analysis to compare differentially expressed genes between oxytetracycline (OXY) susceptible and resistant strains of A. hydrophila. Compared to the A. hydrophila susceptible strain, a total of 22 and 185 genes were differentially expressed in the 4-fold minimal inhibitory concentration (MIC) and 8-fold MIC resistant strains, respectively. Furthermore, the bioinformatics analysis revealed that the sulfur metabolism-related genes were down-regulated. The genes responsible for mannitol metabolism and the efflux pump system were up-regulated in resistant strains, compared to the susceptible strain. Therefore, it suggests that these three pathways may be involved in the OXY resistance evolution in A. hydrophila. The outcome of the transcriptomic data was further validated through quantitative reverse transcription-PCR (qRT-PCR) and Western blot analysis. Overall, the obtained data provides a deeper insight into the intrinsic molecular mechanism of OXY resistance evolution in A. hydrophila

Application of Angiotensin Receptor–Neprilysin Inhibitor in Chronic Kidney Disease Patients: Chinese Expert Consensus

Chronic kidney disease (CKD) is a global public health problem, and cardiovascular disease is the most common cause of death in patients with CKD. The incidence and prevalence of cardiovascular events during the early stages of CKD increases significantly with a decline in renal function. More than 50% of dialysis patients die from cardiovascular disease, including coronary heart disease, heart failure, arrhythmia, and sudden cardiac death. Therefore, developing effective methods to control risk factors and improve prognosis is the primary focus during the diagnosis and treatment of CKD. For example, the SPRINT study demonstrated that CKD drugs are effective in reducing cardiovascular and cerebrovascular events by controlling blood pressure. Uncontrolled blood pressure not only increases the risk of these events but also accelerates the progression of CKD. A co-crystal complex of sacubitril, which is a neprilysin inhibitor, and valsartan, which is an angiotensin receptor blockade, has the potential to be widely used against CKD. Sacubitril inhibits neprilysin, which further reduces the degradation of natriuretic peptides and enhances the beneficial effects of the natriuretic peptide system. In contrast, valsartan alone can block the angiotensin II-1 (AT1) receptor and therefore inhibit the renin–angiotensin–aldosterone system. These two components can act synergistically to relax blood vessels, prevent and reverse cardiovascular remodeling, and promote natriuresis. Recent studies have repeatedly confirmed that the first and so far the only angiotensin receptor–neprilysin inhibitor (ARNI) sacubitril/valsartan can reduce blood pressure more effectively than renin–angiotensin system inhibitors and improve the prognosis of heart failure in patients with CKD. Here, we propose clinical recommendations based on an expert consensus to guide ARNI-based therapeutics and reduce the occurrence of cardiovascular events in patients with CKD

Characterization of acyl-ACP thioesterases for the purpose of diversifying fatty acid synthesis pathway

Acyl-ACP TE selectively hydrolyzes the thiol ester bonds of acyl-ACPs to release free fatty acids, and therefore plays an essential role in determining the output of fatty acid synthesis (FAS) pathway. Comprehensive understanding of acyl-ACP TE is demanded to tailor this biocatalyst for the application in metabolic engineering of FAS pathway. To explore the diversity of acyl-ACP TEs, a total of 31 TEs enzymes were sourced from a wide range of biological taxa, including plants and bacteria, and these were functionally characterized. The results demonstrate that acyl-ACP TEs have great functional diversity relative to the acyl chain length specificity as well as acyl chains that contain additional chemical functionalities. Multiple sequence alignment of plant and bacterial TEs, and structure modeling of CvFatB2 revealed that a previously proposed residue Cys348 is unlikely to be a catalytic residue. Instead, residues Asp309 and Glu347, in addition to previously proposed residues Asn311 and His313 (numbers are based on CvFatB2 sequence), were proposed to be involved in the catalysis of acyl-ACP TEs. In vivo activities of site-directed mutants proved this hypothesis, and a two-step catalytic mechanism for plant and bacterial acyl-ACP TEs is proposed. To identify the region(s) that determine the substrate specificity, two acyl-ACP TEs were used for a domain-shuffling study. Comparing the substrate specificities of the resulting chimeric TEs led to the identification of the most important region that determines the substrate specificity of acyl-ACP TE. Site-directed mutagenesis analysis proved that six residues play critical roles in determining the substrate specificity, including V194 in Fragment II, V217, N223, R226, and R227 in Fragment III, and I268 in Fragment IV. Another three residues, L257, I260, and L289, impact the catalytic activity of acyl-ACP TE, because they are in two proposed ACP binding motifs. A directed evolution approach was successfully developed to improve the fatty acid productivity of acyl-ACP TE. Screening a designed variant library resulted in recovery of TE variants with increased fatty acid productivity and more insight into the relationship between sequences and substrate specificities of acyl-ACP TE.</p

Cardiovascular Adverse Events Associated with Monoclonal Antibody Products in Patients with COVID-19

Little is known about cardiovascular safety profiles for monoclonal antibody products that received the FDA Emergency Use Authorization for COVID-19. In this study, data from the FDA Adverse Event Reporting System from the first quarter of 2020 to the second quarter of 2022 were used to investigate cardiovascular safety signals associated with seven monoclonal antibody products (casirivimab + imdevimab, bamlanivimab, bamlanivimab + etesevimab, sotrovimab, tocilizumab, bebtelovimab, tixagevimab + cilgavimab) in COVID-19 patients. Disproportionality analyses were conducted using reporting odds ratio and information component to identify safety signals. About 10% of adverse events in COVID-19 patients were cardiovascular adverse events. Four monoclonal antibody products (casirivimab + imdevimab, bamlanivimab, bamlanivimab + etesevimab, and bebtelovimab) were associated with higher reporting of hypertension. Tocilizumab was associated with higher reporting of cardiac failure and embolic and thrombotic event. Casirivimab + imdevimab and bamlanivimab were also associated with higher reporting of ischemic heart disease. No cardiovascular safety signals were identified for sotrovimab and tixagevimab + cilgavimab. The results indicate differential cardiovascular safety profiles in monoclonal antibodies. Careful monitoring of cardiovascular events may be considered for certain COVID-19 patients at risk when they are treated with monoclonal antibodies

A Cluster-Weighted Kernel K-Means Method for Multi-View Clustering

Clustering by jointly exploiting information from multiple views can yield better performance than clustering on one single view. Some existing multi-view clustering methods aim at learning a weight for each view to determine its contribution to the final solution. However, the view-weighted scheme can only indicate the overall importance of a view, which fails to recognize the importance of each inner cluster of a view. A view with higher weight cannot guarantee all clusters in this view have higher importance than them in other views. In this paper, we propose a cluster-weighted kernel k-means method for multi-view clustering. Each inner cluster of each view is assigned a weight, which is learned based on the intra-cluster similarity of the cluster compared with all its corresponding clusters in different views, to make the cluster with higher intra-cluster similarity have a higher weight among the corresponding clusters. The cluster labels are learned simultaneously with the cluster weights in an alternative updating way, by minimizing the weighted sum-of-squared errors of the kernel k-means. Compared with the view-weighted scheme, the cluster-weighted scheme enhances the interpretability for the clustering results. Experimental results on both synthetic and real data sets demonstrate the effectiveness of the proposed method

Identification of Active Site Residues Implies a Two-step Catalytic Mechanism for Acyl-ACP Thioesterase

In plants and bacteria that use a Type II fatty acid synthase (FAS), isozymes of acyl-acyl carrier protein (ACP) thioesterase (TEs) hydrolyze the thioester bond of acyl-ACPs, terminating the process of fatty acid biosynthesis. These TEs are therefore critical in determining the fatty acid profiles produced by these organisms. Past characterizations of a limited number of plant-sourced acyl-ACP TEs have suggested a thiol-based, papain-like catalytic mechanism, involving a triad of Cys, His, and Asn residues. In this study, sequence alignment of 1019 plant and bacterial acyl-ACP TEs revealed that the previously proposed Cys catalytic residue is not universally conserved and therefore may not be a catalytic residue. Systematic mutagenesis of this residue to either Ser or Ala in three plant acyl-ACP TEs, CvFatB1 and CvFatB2 from Cuphea viscosissima and CnFatB2 from Cocos nucifera, resulted in enzymatically active variants, demonstrating that this Cys residue (Cys348 in CvFatB2) is not catalytic. In contrast, the multiple sequence alignment, together with the structure modeling of CvFatB2 suggest that the highly conserved Asp309 and Glu347, in addition to previously proposed Asn311 and His313, may be involved in catalysis. The substantial loss of catalytic competence associated with site-directed mutants at these positions confirmed the involvement of these residues in catalysis. By comparing the structures of acyl-ACP TE and the Pseudomonas 4-hydroxybenzoyl-CoA TE, both of which fold in the same hot-dog tertiary structure and catalyze the hydrolysis reaction of thioester bond, we have proposed a two-step catalytic mechanism for acyl-ACP TE that involves an enzyme-bound anhydride intermediate.This is a manuscript of an article published as Jing, Fuyuan, Marna D. Yandeau-Nelson, and Basil J. Nikolau. "Identification of Active Site Residues Implies a Two-step Catalytic Mechanism for Acyl-ACP Thioesterase." Biochemical Journal (2018): doi: 10.1042/BCJ20180470. Posted with permission.</p