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Investigative Ophthalmology & Visual... May 2023Plasmalogens (Plgs) are highly abundant lipids in the retina, and their deficiency leads to severe abnormalities during eye development. The first acylation step in the...
PURPOSE
Plasmalogens (Plgs) are highly abundant lipids in the retina, and their deficiency leads to severe abnormalities during eye development. The first acylation step in the synthesis of Plgs is catalyzed by the enzyme glyceronephosphate O-acyltransferase (GNPAT), which is also known as dihydroxyacetone phosphate-acyltransferase (EC 2.3.1.42). GNPAT deficiency produces rhizomelic chondrodysplasia punctata type 2, a genetic disorder associated with developmental ocular defects. Despite the relevance of retinal Plgs, our knowledge of the mechanisms that regulate their synthesis, and the role of GNPAT during eye development is limited.
METHODS
Using the Xenopus laevis model organism, we characterized by in situ hybridization the expression pattern of gnpat and compared it to glycerol 3-phosphate acyltransferase mitochondrial (gpam or gpat1) during eye neurogenesis, lamination, and morphogenesis. The Xenopus Gnpat was biochemically characterized in a heterologous expression system in yeast.
RESULTS
During development, gnpat is expressed in proliferative cells of the retina and lens, and post-embryogenesis in proliferative cells of the ciliary marginal zone and lens epithelium. In contrast, gpam expression is mainly restricted to photoreceptors. Xenopus Gnpat expressed in yeast is present in both soluble and membrane fractions, but only the membrane-bound enzyme displays activity. The amino terminal of Gnpat, conserved in humans, shows lipid binding capacity that is enhanced by phosphatidic acid.
CONCLUSIONS
Enzymes involved in the Plgs and glycerophospholipid biosynthetic pathways are differentially expressed during eye morphogenesis. The gnpat expression pattern and the molecular determinants regulating Gnpat activity advance our knowledge of this enzyme, contributing to our understanding of the retinal pathophysiology associated with GNPAT deficiency.
Topics: Animals; Humans; Acyltransferases; Plasmalogens; Saccharomyces cerevisiae; Xenopus laevis; Xenopus Proteins
PubMed: 37204785
DOI: 10.1167/iovs.64.5.17 -
Molecules (Basel, Switzerland) Apr 2021Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin...
Anthocyanins are pigments with appealing hues that are currently being used as sources of natural colorants. The interaction of acylation on the stability of anthocyanin molecules has long been known. Maize is an abundant source of malonylglucoside and dimalonylglucoside anthocyanins. The enzyme Aat1 is an anthocyanin acyltransferase known to synthesize the majority of acylated anthocyanins in maize. In this paper, we characterize the substrate specificity and reaction kinetics of Aat1. It was found that Aat1 has anthocyanin 3--glucoside dimalonyltransferase activity and is only the second enzyme of this type characterized to this date. Our results indicate that Aat1 can utilize malonyl-CoA; succinyl-CoA and every anthocyanin 3--glucoside tested. Results of this study provide insight into the structure-function relations of dimalonyltransferases and give a unique insight into the activity of monocot anthocyanin acyltransferases.
Topics: Acyltransferases; Anthocyanins; Chromatography, High Pressure Liquid; Enzyme Activation; Kinetics; Mass Spectrometry; Molecular Structure; Recombinant Proteins; Structure-Activity Relationship; Substrate Specificity; Zea mays
PubMed: 33916241
DOI: 10.3390/molecules26072020 -
The Journal of Biological Chemistry Apr 2023Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to...
Transferases are ubiquitous across all known life. While much work has been done to understand and describe these essential enzymes, there have been minimal efforts to exert tight and reversible control over their activity for various biotechnological applications. Here, we apply a rational, computation-guided methodology to design and test a transferase-class enzyme allosterically regulated by light-oxygen-voltage 2 sensing domain. We utilize computational techniques to determine the intrinsic allosteric networks within N-acyltransferase (Orf11/∗Dbv8) and identify potential allosteric sites on the protein's surface. We insert light-oxygen-voltage 2 sensing domain at the predicted allosteric site, exerting reversible control over enzymatic activity. We demonstrate blue-light regulation of N-acyltransferase (Orf11/∗Dbv8) function. Our study for the first time demonstrates optogenetic regulation of a transferase-class enzyme as a proof-of-concept for controllable transferase design. This successful design opens the door for many future applications in metabolic engineering and cellular programming.
Topics: Acyltransferases; Allosteric Regulation; Allosteric Site; Light; Oxygen; Protein Domains; Recombinant Fusion Proteins; Enzyme Activation
PubMed: 36841477
DOI: 10.1016/j.jbc.2023.103069 -
Acta Crystallographica. Section F,... Sep 2021Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of...
Acyltransferases are responsible for the selection and loading of acyl units onto carrier proteins in polyketide and fatty-acid biosynthesis. Despite the importance of protein-protein interactions between the acyltransferase and the carrier protein, structural information on acyltransferase-carrier protein interactions is limited because of the transient interactions between them. In the biosynthesis of the polyketide vicenistatin, the acyltransferase VinK recognizes the carrier protein VinL for the transfer of a dipeptidyl unit. The crystal structure of a VinK-VinL covalent complex formed with a 1,2-bismaleimidoethane cross-linking reagent has been determined previously. Here, the crystal structure of a VinK-VinL covalent complex formed with a pantetheine cross-linking probe is reported at 1.95 Å resolution. In the structure of the VinK-VinL-probe complex, the pantetheine probe that is attached to VinL is covalently connected to the side chain of the mutated Cys106 of VinK. The interaction interface between VinK and VinL is essentially the same in the two VinK-VinL complex structures, although the position of the pantetheine linker slightly differs. This structural observation suggests that interface interactions are not affected by the cross-linking strategy used.
Topics: Acyltransferases; Cross-Linking Reagents; Crystallography, X-Ray; Mutagenesis, Site-Directed; Mutation; Pantetheine; Protein Conformation; Protein Interaction Domains and Motifs; Substrate Specificity
PubMed: 34473106
DOI: 10.1107/S2053230X21008761 -
Biochemical Society Transactions Apr 2015The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular... (Review)
Review
The post-translational modification of proteins with lipid moieties confers spatial and temporal control of protein function by restricting their subcellular distribution or movement in the extracellular milieu. Yet, little is known about the significance of lipid selectivity to the activity of proteins targeted for such modifications. Membrane bound O-acyl transferases (MBOATs) are a superfamily of multipass enzymes that transfer fatty acids on to lipid or protein substrates. Three MBOATs constitute a subfamily with secreted signalling molecules for substrates, the Wnt, Hedgehog (Hh) and Ghrelin proteins. Given their important roles in adult tissue homoeostasis, all three molecules and their respective associated acyltransferases provide a framework for interrogating the role of extracellular acylation events in cell-to-cell communication. Here, we discuss how the preference for a fatty acyl donor in the Wnt acyltransferase porcupine (Porcn) and possibly in other protein lipidation enzymes may provide a means for coupling metabolic health at the single cell level to communal cell fate decision-making in complex multicellular organisms.
Topics: Acylation; Acyltransferases; Animals; Cell Communication; Cell Differentiation; Cell Membrane; Fatty Acids; Humans; Membrane Proteins; Protein Processing, Post-Translational; Substrate Specificity; Wnt Signaling Pathway
PubMed: 25849923
DOI: 10.1042/BST20140282 -
Angewandte Chemie (International Ed. in... Jan 2021Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor...
Promiscuous acyltransferase activity is the ability of certain hydrolases to preferentially catalyze acyl transfer over hydrolysis, even in bulk water. However, poor enantioselectivity, low transfer efficiency, significant product hydrolysis, and limited substrate scope represent considerable drawbacks for their application. By activity-based screening of several hydrolases, we identified the family VIII carboxylesterase, EstCE1, as an unprecedentedly efficient acyltransferase. EstCE1 catalyzes the irreversible amidation and carbamoylation of amines in water, which enabled the synthesis of the drug moclobemide from methyl 4-chlorobenzoate and 4-(2-aminoethyl)morpholine (ca. 20 % conversion). We solved the crystal structure of EstCE1 and detailed structure-function analysis revealed a three-amino acid motif important for promiscuous acyltransferase activity. Introducing this motif into an esterase without acetyltransferase activity transformed a "hydrolase" into an "acyltransferase".
Topics: Acyltransferases; Carboxylic Ester Hydrolases; Catalysis; Hydrolysis; Hydrophobic and Hydrophilic Interactions; Proof of Concept Study; Structure-Activity Relationship; Substrate Specificity
PubMed: 33140887
DOI: 10.1002/anie.202014169 -
Comparative Biochemistry and... Mar 2010BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the... (Comparative Study)
Comparative Study
Comparative genomics and proteomics of vertebrate diacylglycerol acyltransferase (DGAT), acyl CoA wax alcohol acyltransferase (AWAT) and monoacylglycerol acyltransferase (MGAT).
BLAT (BLAST-Like Alignment Tool) analyses of the opossum (Monodelphis domestica) and zebrafish (Danio rerio) genomes were undertaken using amino acid sequences of the acylglycerol acyltransferase (AGAT) superfamily. Evidence is reported for 8 opossum monoacylglycerol acyltransferase-like (MGAT) (E.C. 2.3.1.22) and diacylglycerol acyltransferase-like (DGAT) (E.C. 2.3.1.20) genes and proteins, including DGAT1, DGAT2, DGAT2L6 (DGAT2-like protein 6), AWAT1 (acyl CoA wax alcohol acyltransferase 1), AWAT2, MGAT1, MGAT2 and MGAT3. Three of these genes (AWAT1, AWAT2 and DGAT2L6) are closely localized on the opossum X chromosome. Evidence is also reported for six zebrafish MGAT- and DGAT-like genes, including two DGAT1-like genes, as well as DGAT2-, MGAT1-, MGAT2- and MGAT3-like genes and proteins. Predicted primary, secondary and transmembrane structures for the opossum and zebrafish MGAT-, AWAT- and DGAT-like subunits and the intron-exon boundaries for genes encoding these enzymes showed a high degree of similarity with other members of the AGAT superfamily, which play major roles in triacylglyceride (DGAT), diacylglyceride (MGAT) and wax ester (AWAT) biosynthesis. Alignments of predicted opossum, zebrafish and other vertebrate DGAT1, DGAT2, other DGAT2-like and MGAT-like amino acid sequences with known human and mouse enzymes demonstrated conservation of residues which are likely to play key roles in catalysis, lipid binding or in maintaining structure. Phylogeny studies of the human, mouse, opossum, zebrafish and pufferfish MGAT- and DGAT-like enzymes indicated that the common ancestors for these genes predated the appearance of bony fish during vertebrate evolution whereas the AWAT- and DGAT2L6-like genes may have appeared more recently prior to the appearance of marsupial and eutherian mammals.
Topics: Acyltransferases; Amino Acid Sequence; Animals; Diacylglycerol O-Acyltransferase; Exons; Genetic Variation; Humans; Isoenzymes; Mice; Molecular Sequence Data; Opossums; Phylogeny; Protein Structure, Secondary; Protein Subunits; Proteomics; RNA, Messenger; Sequence Alignment; Vertebrates; Zebrafish
PubMed: 20374941
DOI: 10.1016/j.cbd.2009.09.004 -
Angewandte Chemie (International Ed. in... Jul 2020Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are...
Certain hydrolases preferentially catalyze acyl transfer over hydrolysis in an aqueous environment. However, the molecular and structural reasons for this phenomenon are still unclear. Herein, we provide evidence that acyltransferase activity in esterases highly correlates with the hydrophobicity of the substrate-binding pocket. A hydrophobicity scoring system developed in this work allows accurate prediction of promiscuous acyltransferase activity solely from the amino acid sequence of the cap domain. This concept was experimentally verified by systematic investigation of several homologous esterases, leading to the discovery of five novel promiscuous acyltransferases. We also developed a simple yet versatile colorimetric assay for rapid characterization of novel acyltransferases. This study demonstrates that promiscuous acyltransferase activity is not as rare as previously thought and provides access to a vast number of novel acyltransferases with diverse substrate specificity and potential applications.
Topics: Acyltransferases; Amino Acid Sequence; Catalysis; High-Throughput Screening Assays; Hydrolases; Hydrolysis; Hydrophobic and Hydrophilic Interactions
PubMed: 32243661
DOI: 10.1002/anie.202003635 -
American Journal of Physiology.... Jul 2009Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of... (Review)
Review
Monoacyglycerol acyltransferases (MGATs) and diacylglycerol acyltransferases (DGATs) catalyze two consecutive steps of enzyme reactions in the synthesis of triacylglycerols (TAGs). The metabolic complexity of TAG synthesis is reflected by the presence of multiple isoforms of MGAT and DGAT enzymes that differ in catalytic properties, subcellular localization, tissue distribution, and physiological functions. MGAT and DGAT enzymes play fundamental roles in the metabolism of monoacylglycerol (MAG), diacylglycerol (DAG), and triacylglycerol (TAG) that are involved in many aspects of physiological functions, such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, signal transduction, satiety, and lactation. The recent progress in the phenotypic characterization of mice deficient in MGAT and DGAT enzymes and the development of chemical inhibitors have revealed important roles of these enzymes in the regulation of energy homeostasis and insulin sensitivity. Consequently, selective inhibition of MGAT or DGAT enzymes by synthetic compounds may provide novel treatment for obesity and its related metabolic complications.
Topics: Acyltransferases; Animals; Diacylglycerol O-Acyltransferase; Drug Delivery Systems; Energy Metabolism; Humans; Immunoglobulins; Models, Biological; Obesity; Triglycerides
PubMed: 19116371
DOI: 10.1152/ajpendo.90949.2008 -
International Journal of Molecular... Mar 2021The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various...
The glycine conjugation pathway in humans is involved in the metabolism of natural substrates and the detoxification of xenobiotics. The interactions between the various substrates in this pathway and their competition for the pathway enzymes are currently unknown. The pathway consists of a mitochondrial xenobiotic/medium-chain fatty acid: coenzyme A (CoA) ligase (ACSM2B) and glycine -acyltransferase (GLYAT). The catalytic mechanism and substrate specificity of both of these enzymes have not been thoroughly characterised. In this study, the level of evolutionary conservation of GLYAT missense variants and haplotypes were analysed. From these data, haplotype variants were selected (156Asn > Ser, [17Ser > Thr,156Asn > Ser] and [156Asn > Ser,199Arg > Cys]) in order to characterise the kinetic mechanism of the enzyme over a wide range of substrate concentrations. The 156Asn > Ser haplotype has the highest frequency and the highest relative enzyme activity in all populations studied, and hence was used as the reference in this study. Cooperative substrate binding was observed, and the kinetic data were fitted to a two-substrate Hill equation. The coding region of the gene was found to be highly conserved and the rare 156Asn > Ser,199Arg > Cys variant negatively affected the relative enzyme activity. Even though the 156Asn > Ser,199Arg > Cys variant had a higher affinity for benzoyl-CoA ( = 61.2 µM), was reduced to 9.8% of the most abundant haplotype 156Asn > Ser ( = 96.6 µM), while the activity of 17Ser > Thr,156Asn > Ser ( = 118 µM) was 73% of 156Asn > Ser. The in vitro kinetic analyses of the effect of the 156Asn > Ser,199Arg > Cys variant on human GLYAT enzyme activity indicated that individuals with this haplotype might have a decreased ability to metabolise benzoate when compared to individuals with the 156Asn > Ser variant. Furthermore, the accumulation of acyl-CoA intermediates can inhibit ACSM2B leading to a reduction in mitochondrial energy production.
Topics: Acyl Coenzyme A; Acyltransferases; Animals; Conserved Sequence; Gene Frequency; Glycine; Humans; Kinetics; Mutation; Phylogeny
PubMed: 33803916
DOI: 10.3390/ijms22063129