Dataset Information

Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity.

ABSTRACT: Plant glycoproteins display a characteristic type of O-glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cells using the baculovirus expression system and applied to synthetic peptides representing the O-glycosylated region of erythropoietin (EPO), IgA1, Art v 1 and the Arabidopsis thaliana glycoprotein STRUBBELIG. Unlike the situation in the moss Physcomitrella patens, where one particular P4H was mainly responsible for the oxidation of erythropoietin, the tobacco P4Hs exhibited rather similar activities, albeit with biased substrate preferences and preferred sites of oxidation. From a biotechnological viewpoint, this result means that silencing/knockout of a single P4H in N. benthamiana cannot be expected to result in the abolishment of the plant-specific oxidation of prolyl residues in a recombinant protein.

SUBMITTER: Mocsai R

PROVIDER: S-EPMC7960765 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity.

Mócsai Réka R Göritzer Kathrin K Stenitzer David D Maresch Daniel D Strasser Richard R Altmann Friedrich F

Frontiers in plant science 20210302

Plant glycoproteins display a characteristic type of O-glycosylation where short arabinans or larger arabinogalactans are linked to hydroxyproline. The conversion of proline to 4-hydroxyproline is accomplished by prolyl-hydroxylases (P4Hs). Eleven putative Nicotiana benthamiana P4Hs, which fall in four homology groups, have been identified by homology searches using known Arabidopsis thaliana P4H sequences. One member of each of these groups has been expressed in insect cell ...[more]

PMID: 33737944

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Project description:Glucosinolates (GLSs) are amino acid-derived defense compounds characteristic of the Brassicales order. Cytochromes P450s of the CYP79 family are the entry point into the biosynthetic pathway of the GLS core structure and catalyze the conversion of amino acids to oximes. In Arabidopsis thaliana, CYP79A2, CYP79B2, CYP79B3, CYP79F1, and CYP79F2 have been functionally characterized and are responsible for the biosynthesis of phenylalanine-, tryptophan-, and methionine-derived GLSs, respectively. However, the substrate(s) for CYP79C1 and CYP79C2 were unknown. Here, we investigated the function of CYP79C1 and CYP79C2 by transiently co-expressing the genes together with three sets of remaining genes required for GLS biosynthesis in Nicotiana benthamiana. Co-expression of CYP79C2 with either the aliphatic or aromatic core structure pathways resulted in the production of primarily leucine-derived 2-methylpropyl GLS and phenylalanine-derived benzyl GLS, along with minor amounts of GLSs from isoleucine, tryptophan, and tyrosine. Co-expression of CYP79C1 displayed minor amounts of GLSs from valine, leucine, isoleucine, and phenylalanine with the aliphatic core structure pathway, and similar GLS profile (except the GLS from valine) with the aromatic core structure pathway. Additionally, we co-expressed CYP79C1 and CYP79C2 with the chain elongation and aliphatic core structure pathways. With the chain elongation pathway, CYP79C2 still mainly produced 2-methylpropyl GLS derived from leucine, accompanied by GLSs derived from isoleucine and from chain-elongated mono- and dihomoleucine, but not from phenylalanine. However, co-expression of CYP79C1 only resulted in GLSs derived from chain-elongated amino acid substrates, dihomoleucine and dihomomethionine, when the chain elongation pathway was present. This shows that CYP79 activity depends on the specific pathways co-expressed and availability of amino acid precursors, and that description of GLS core structure pathways as "aliphatic" and "aromatic" pathways is not suitable, especially in an engineering context. This is the first characterization of members of the CYP79C family. Co-expression of CYP79 enzymes with engineered GLS pathways in N. benthamiana is a valuable tool for simultaneous testing of substrate specificity against multiple amino acids.

Project description:Bacteria are the principal consumers of dissolved organic carbon (DOC) in the ocean and predation of bacteria makes organic carbon available to higher trophic levels. The efficiency with which bacteria convert the consumed carbon (C) into biomass (i.e., carbon growth efficiency, Y) determines their ecological as well as biogeochemical role in marine ecosystems. Yet, it is still unclear how changes in temperature will affect Y and, hence, the transfer of consumed C to higher trophic levels. Here, we experimentally investigated the effect of temperature on metabolic functions of coastal microbial communities inoculated in both nutrient-limited chemostats and nutrient-unlimited turbidostats. We inoculated chemostats and turbidostats with coastal microbial communities into seawater culture medium augmented with 20 and 100 μmol L-1 of glucose respectively and measured CO2 production, carbon biomass and cell abundance. Chemostats were cultured between 14 and 26°C and specific growth rates (μ) between 0.05 and 6.0 day-1, turbidostats were cultured between 10 and 26°C with specific growth rates ranging from 28 to 62 day-1. In chemostats under substrate limitation, which is common in the ocean, the specific respiration rate (r, day-1) showed no trend with temperature and was roughly proportional to μ, implying that carbon growth efficiency (Y) displayed no tendency with temperature. The response was very different in turbidostats under temperature-limited, nutrient-repleted growth, here μ increased with temperature but r decreased resulting in an increase of Y with temperature (Q10: 2.6). Comparison of our results with data from the literature on the respiration rate and cell weight of monospecific bacteria indicates that in general the literature data behaved similar to chemostat data, showing no trend in specific respiration with temperature. We conclude that respiration rates of nutrient-limited bacteria measured at a certain temperature cannot be adjusted to different temperatures with a temperature response function similar to Q10 or Arrhenius. However, the cellular respiration rate and carbon demand rate (both: mol C cell-1 day-1) show statistically significant relations with cellular carbon content (mol C cell-1) in chemostats, turbidostats, and the literature data.

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Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity.

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Prolyl Hydroxylase Paralogs in <i>Nicotiana benthamiana</i> Show High Similarity With Regard to Substrate Specificity.

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Prolyl Hydroxylase Paralogs in Nicotiana benthamiana Show High Similarity With Regard to Substrate Specificity.

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Prolyl Hydroxylase Paralogs in &lt;i&gt;Nicotiana benthamiana&lt;/i&gt; Show High Similarity With Regard to Substrate Specificity.

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OmicsDI is part of the ELIXIR infrastructure

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Prolyl Hydroxylase Paralogs in <i>Nicotiana benthamiana</i> Show High Similarity With Regard to Substrate Specificity.