Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga Isochrysis sp.

2017 ◽  
Vol 40 (3) ◽  
pp. 577-584 ◽  
Author(s):  
S. Thiyagarajan ◽  
M. Arumugam ◽  
N. Senthil ◽  
S. Vellaikumar ◽  
S. Kathiresan
Keyword(s):  
Substrate Specificity ◽  
Functional Characterization ◽  
Marine Microalga ◽  
Δ6 Desaturase

scholarly journals Substrate specificity and preference of Δ6-desaturase ofMucor rouxii

FEBS Letters ◽  
2005 ◽  
Vol 579 (12) ◽  
pp. 2744-2748 ◽  
Author(s):  
Sutthicha Na-Ranong ◽  
Kobkul Laoteng ◽  
Prasat Kittakoop ◽  
Morakot Tantichareon ◽  
Supapon Cheevadhanarak
Keyword(s):  
Substrate Specificity ◽  
Δ6 Desaturase

scholarly journals The Sequence and a Three-Dimensional Structural Analysis Reveal Substrate Specificity Among Snake Venom Phosphodiesterases

Toxins ◽  
2019 ◽  
Vol 11 (11) ◽  
pp. 625 ◽  
Author(s):  
Ullah ◽  
Ullah ◽  
Ali ◽  
Betzel ◽  
ur Rehman
Keyword(s):  
Substrate Specificity ◽  
Snake Venom ◽  
Model Building ◽  
Function Analysis ◽  
Functional Characterization ◽  
3D Structure ◽  
Three Dimensional ◽  
High Sequence Identity ◽  
Sequence Comparisons ◽  
Specific Domain

(1) Background. Snake venom phosphodiesterases (SVPDEs) are among the least studied venom enzymes. In envenomation, they display various pathological effects, including induction of hypotension, inhibition of platelet aggregation, edema, and paralysis. Until now, there have been no 3D structural studies of these enzymes, thereby preventing structure–function analysis. To enable such investigations, the present work describes the model-based structural and functional characterization of a phosphodiesterase from Crotalus adamanteus venom, named PDE_Ca. (2) Methods. The PDE_Ca structure model was produced and validated using various software (model building: I-TESSER, MODELLER 9v19, Swiss-Model, and validation tools: PROCHECK, ERRAT, Molecular Dynamic Simulation, and Verif3D). (3) Results. The proposed model of the enzyme indicates that the 3D structure of PDE_Ca comprises four domains, a somatomedin B domain, a somatomedin B-like domain, an ectonucleotide pyrophosphatase domain, and a DNA/RNA non-specific domain. Sequence and structural analyses suggest that differences in length and composition among homologous snake venom sequences may account for their differences in substrate specificity. Other properties that may influence substrate specificity are the average volume and depth of the active site cavity. (4) Conclusion. Sequence comparisons indicate that SVPDEs exhibit high sequence identity but comparatively low identity with mammalian and bacterial PDEs.

Functional characterization, localization, and molecular identification of rabbit intestinal N-amino acid transporter

2008 ◽  
Vol 294 (6) ◽  
pp. G1301-G1310 ◽  
Author(s):  
Jamilur R. Talukder ◽  
Ramesh Kekuda ◽  
Prosenjit Saha ◽  
Puttur D. Prasad ◽  
Vadivel Ganapathy ◽  
...  
Keyword(s):  
Amino Acid ◽  
Substrate Specificity ◽  
Expression System ◽  
Functional Characterization ◽  
Membrane Vesicles ◽  
Crypt Cell ◽  
Hill Coefficient ◽  
Reading Frame ◽  
Glutamine Transporter ◽  
Crypt Cells

We have characterized the Na-glutamine cotransporter in the rabbit intestinal crypt cell brush border membrane vesicles (BBMV). Substrate specificity experiments showed that crypt cell glutamine uptake is mediated by system N. Real-time PCR experiments showed that SN2 (SLC38A5) mRNA is more abundant in crypt cells compared with SN1 (SLC38A3), indicating that SN2 is the major glutamine transporter present in the apical membrane of the crypt cells. SN2 cDNA was obtained by screening a rabbit intestinal cDNA library with human SN1 used as probe. Rabbit SN2 cDNA encompassed a 473-amino-acid-long open reading frame. SN2 protein displayed 87% identity and 91% similarity to human SN2. Functional characterization studies of rabbit SN2 were performed by using vaccinia virus-mediated transient expression system. Substrate specificity of the cloned transporter was identical to that of SN2 described in the literature and matched well with substrate specificity experiments performed using crypt cell BBMV. Cloned rabbit SN2, analogous to its human counterpart, is Li+ tolerant. Hill coefficient for Li+ activation of rabbit SN2-mediated uptake was 1. Taken together, functional data from the crypt cell BBMV and the cloned SN2 cDNA indicate that the crypt cell glutamine transport is most likely mediated by SN2.

scholarly journals Functional Characterization of the Type III Secretion Substrate Specificity Switch Protein HpaC from Xanthomonas campestris pv. vesicatoria

2011 ◽  
Vol 79 (8) ◽  
pp. 2998-3011 ◽  
Author(s):  
Steve Schulz ◽  
Daniela Büttner
Keyword(s):  
Amino Acids ◽  
Substrate Specificity ◽  
Type Iii Secretion ◽  
Protein Function ◽  
Xanthomonas Campestris ◽  
Functional Characterization ◽  
Effector Proteins ◽  
Content Type ◽  
Type Iii ◽  
In The Wild

ABSTRACTPathogenicity ofXanthomonas campestrispv.vesicatoriadepends on a type III secretion (T3S) system which translocates effector proteins into eukaryotic cells and is associated with an extracellular pilus and a translocon in the host plasma membrane. T3S substrate specificity is controlled by the cytoplasmic switch protein HpaC, which interacts with the C-terminal domain of the inner membrane protein HrcU (HrcUC). HpaC promotes the secretion of translocon and effector proteins but prevents the efficient secretion of the early T3S substrate HrpB2, which is required for pilus assembly. In this study, complementation assays with serial 10-amino-acid HpaC deletion derivatives revealed that the T3S substrate specificity switch depends on N- and C-terminal regions of HpaC, whereas amino acids 42 to 101 appear to be dispensable for the contribution of HpaC to the secretion of late substrates. However, deletions in the central region of HpaC affect the secretion of HrpB2, suggesting that the mechanisms underlying HpaC-dependent control of early and late substrates can be uncoupled. The results of interaction and expression studies with HpaC deletion derivatives showed that amino acids 112 to 212 of HpaC provide the binding site for HrcUCand severely reduce T3S when expressed ectopically in the wild-type strain. We identified a conserved phenylalanine residue at position 175 of HpaC that is required for both protein function and the binding of HpaC to HrcUC. Taking these findings together, we concluded that the interaction between HpaC and HrcUCis essential but not sufficient for T3S substrate specificity switching.

scholarly journals Functional Characterization of an Anthocyanin Dimalonyltransferase in Maize

Molecules ◽  
2021 ◽  
Vol 26 (7) ◽  
pp. 2020
Author(s):  
Michael Paulsmeyer ◽  
John Juvik
Keyword(s):  
Substrate Specificity ◽  
Structure Function ◽  
Functional Characterization ◽  
Malonyl Coa ◽  
Natural Colorants ◽  
Acylated Anthocyanins ◽  
The Stability ◽  
Kinetics Of ◽  
Insight Into

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-O-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-O-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.

scholarly journals Plastidic Δ6 fatty-acid desaturases with distinctive substrate specificity regulate the pool of c18-pufas in the ancestral picoalga ostreococcus tauri

2020 ◽  
Author(s):  
Charlotte Degraeve-Guilbault C. ◽  
Rodrigo E. Gomez ◽  
Cécile. Lemoigne ◽  
Nattiwong Pankansem ◽  
Soizic Morin ◽  
...  
Keyword(s):  
Substrate Specificity ◽  
Acyl Chain ◽  
Cytochrome B5 ◽  
Head Group ◽  
Fatty Acid Desaturases ◽  
Physiological Alterations ◽  
Lipid Head Group ◽  
Species Specific ◽  
Native Host ◽  
Δ6 Desaturase

ABSTRACTEukaryotic Δ6-desaturases are microsomal enzymes which balance the synthesis of ω-3 and ω-6 C18-polyunsaturated-fatty-acids (PUFA) accordingly to their specificity. In several microalgae, including O. tauri, plastidic C18-PUFA are specifically regulated by environmental cues suggesting an autonomous control of Δ6-desaturation of plastidic PUFA. Sequence retrieval from O. tauri desaturases, highlighted two putative Δ6/Δ8-desaturases sequences clustering, with other microalgal homologs, apart from other characterized Δ-6 desaturases. Their overexpression in heterologous hosts, including N. benthamiana and Synechocystis, unveiled their Δ6-desaturation activity and plastid localization. O. tauri lines overexpressing these Δ6-desaturases no longer adjusted their plastidic C18-PUFA amount under phosphate starvation but didn’t show any obvious physiological alterations. Detailed lipid analyses from the various overexpressing hosts, unravelled that the substrate features involved in the Δ6-desaturase specificity importantly involved the lipid head-group and likely the non-substrate acyl-chain, in addition to the overall preference for the ω-class of the substrate acyl-chain. The most active desaturase displayed a broad range substrate specificity for plastidic lipids and a preference for ω-3 substrates, while the other was selective for ω-6 substrates, phosphatidylglycerol and 16:4-galactolipid species specific to the native host. The distribution of plastidial Δ6-desaturase products in eukaryotic hosts suggested the occurrence of C18-PUFA export from the plastid.One sentence summaryOsteococcus tauri plastidic lipid C18-PUFA remodelling involves two plastid-located cytochrome-b5 fused Δ6-desaturases with distinct preferences for both head-group and acyl-chain.

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