Expression and Purification of Cp3GT: Structural Analysis and Modeling of a Key Plant Flavonol-3-O Glucosyltransferase from Citrus paradisi.

Glycosyltransferases (GTs) are pivotal enzymes in the biosynthesis of various biological molecules. This study focuses on the scale-up, expression, and purification of a plant flavonol-specific 3-O glucosyltransferase (Cp3GT), a key enzyme from Citrus paradisi, for structural analysis and modeling. The challenges associated with recombinant protein production in Pichia pastoris, such as proteolytic degradation, were addressed through the optimization of culture conditions and purification processes. The purification strategy employed affinity, anion exchange, and size exclusion chromatography, leading to greater than 95% homogeneity for Cp3GT. In silico modeling, using D-I-TASSER and COFACTOR integrated with the AlphaFold2 pipeline, provided insights into the structural dynamics of Cp3GT and its ligand binding sites, offering predictions for enzyme-substrate interactions. These models were compared to experimentally derived structures, enhancing understanding of the enzyme's functional mechanisms. The findings present a comprehensive approach to produce a highly purified Cp3GT which is suitable for crystallographic studies and to shed light on the structural basis of flavonol specificity in plant GTs. The significant implications of these results for synthetic biology and enzyme engineering in pharmaceutical applications are also considered.

The Effect of Recombinant Tags on Citrus paradisi Flavonol-Specific 3-O Glucosyltransferase Activity.

Recombinant tags are used extensively in protein expression systems to allow purification through IMAC (Immobilized Metal Affinity Chromatography), identification through Western blot, and to facilitate crystal formation for structural analysis. While widely used, their role in enzyme characterization has raised concerns with respect to potential impact on activity. In this study, a flavonol-specific 3-O glucosyltransferase (Cp3GT) from grapefruit (Citrus paradisi) was expressed in Pichia pastoris, and was assayed in its untagged form and with a C-terminal c-myc/6x His tag under various conditions to determine the effect of tags. Prior characterization of pH optima for Cp3GT obtained through expression in Escherichia coli, containing an N-terminal thioredoxin/6x His tag, indicated an optimal pH of 7-7.5, which is indicative of a normal physiological pH and agrees with other glucosyltransferase (GT) pH optima. However, characterization of Cp3GT expressed using P. pastoris with a C-terminal c-myc-6x His tag showed a higher optimal pH of 8.5-9. This suggests a possible tag effect or an effect related to physiological differences between the cell expression systems. Results testing recombinant Cp3GT expressed in Pichia with and without C-terminal tags showed a possible tag effect with regard to substrate preference and interactions with metals, but no apparent effect on enzymatic kinetics or pH optima.

Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi.

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

Secondary product glucosyltransferase and putative glucosyltransferase expression during Citrus paradisi (c.v. Duncan) growth and development.

Flavonoids are secondary metabolites that have significant roles in plant defense and human nutrition. Glucosyltransferases (GTs) catalyze the transfer of sugars from high energy sugar donors to other substrates. Several different secondary product GTs exist in the tissues of grapefruit making it a model plant for studying their structure and function. The goal of this investigation was to determine the expression patterns of seven putative secondary product GTs during grapefruit growth and development by quantifying mRNA expression levels in the roots, stems, leaves, flowers, and mature fruit to establish whether the genes are expressed constitutively or if one or more could be expressed in a tissue specific manner and/or developmentally regulated. Six growth stages were defined from which RNA was extracted, and expression levels were quantified by standardized densitometry of gene-specific RT-PCR products. Results show that there were variable degrees of PGT expression in different tissues and at different developmental stages. These results add to the growing knowledge base of dynamics of expression and potential regulation of secondary metabolism in Citrus paradisi.

Identification, recombinant expression, and biochemical characterization of a flavonol 3-O-glucosyltransferase clone from Citrus paradisi.

Glucosylation is a predominant flavonoid modification reaction affecting the solubility, stability, and subsequent bioavailability of these metabolites. Flavonoid glycosides affect taste characteristics in citrus making the associated glucosyltransferases particularly interesting targets for biotechnology applications in these species. In this work, a Citrus paradisi glucosyltransferase gene was identified, cloned, and introduced into the pET recombinant protein expression system utilizing primers designed against a predicted flavonoid glucosyltransferase gene (AY519364) from Citrus sinensis. The encoded C. paradisi protein is 51.2 kDa with a predicted pI of 6.27 and is 96% identical to the C. sinensis homologue. A number of compounds from various flavonoid subclasses were tested, and the enzyme glucosylated only the flavonol aglycones quercetin (K(m)(app)=67 microM; V(max)=20.45 pKat/microg), kaempferol (K(m)(app)=12 microM; V(max)=11.63 pKat/microg), and myricetin (K(m)(app)=33 microM; V(max)=12.21 pKat/microg) but did not glucosylate the anthocyanidin, cyanidin. Glucosylation occurred at the 3 hydroxyl position as confirmed by HPLC and TLC analyses with certified reference compounds. The optimum pH was 7.5 with a pronounced buffer effect noted for reactions performed in Tris-HCl buffer. The enzyme was inhibited by Cu(2+), Fe(2+), and Zn(2+) as well as UDP (K(i)(app)=69.5 microM), which is a product of the reaction. Treatment of the enzyme with a variety of amino acid modifying compounds suggests that cysteine, histidine, arginine, tryptophan, and tyrosine residues are important for activity. The thorough characterization of this C. paradisi flavonol 3-O-glucosyltransferase adds to the growing base of glucosyltransferase knowledge, and will be used to further investigate structure-function relationships.

Flavanone 3-hydroxylase expression in Citrus paradisi and Petunia hybrida seedlings.

Petunia hybrida and Citrus paradisi have significantly different flavonoid accumulation patterns. Petunia sp. tend to accumulate flavonol glycosides and anthocyanins while Citrus paradisi is known for its accumulation of flavanone diglycosides. One possible point of regulation of flavanone metabolism is flavanone 3-hydroxylase (F3H) expression. To test whether this is a key factor in the different flavanone usage by Petunia hybrida and Citrus paradisi, F3H mRNA expression in seedlings of different developmental stages was measured using semi-quantitative RT-PCR. Primers were designed to conserved regions of F3H and used to amplify an approximately 350 bp segment for quantitation by PhosphorImaging. Primary leaves of 32 day old grapefruit seedlings and a grapefruit flower bud had the highest levels of F3H mRNA expression. Petunia seedlings had much lower levels of F3H mRNA expression relative to grapefruit. The highest expression in petunia was in primary leaves and roots of 65 day old seedlings. These results indicate that preferential use of naringenin for production of high levels of flavanone glycosides in young grapefruit leaves cannot be attributed to decreased F3H mRNA expression.

Quantification of the production of dihydrokaempferol by flavanone 3-hydroxytransferase using capillary electrophoresis.

A sensitive method using capillary electrophoresis for the separation, detection, and quantification of dihydrokaempferol (1) is reported. Well-resolved, sharp symmetrical peaks were obtained in grapefruit leaf extracts for 1, naringenin (2), and the internal standard, naringin (3). Long columns were required to resolve 1 from 2 in crude enzyme reactions and this resulted in run times of 60 min. The limit of detection for 1 was found to be 1.44 ng/microL (4.2 pg). The method showed excellent linearity and reproducibility. The method was used to determine the activity of flavanone 3-hydroxytransferase (F3H) in leaf tissue of grapefruit by quantification of the production of dihydrokaempferol in controlled time course reactions. The sensitivity of the method makes it adaptable to assaying F3H activity in individual young seedlings and/or in small tissue samples and requires only 100 mg of tissue.