Identification, Recombinant Expression, and Biochemical Analysis of

Feb 18, 2016 - ... EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST ...
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Identification, Recombinant Expression, and Biochemical Analysis of Putative Secondary Product Glucosyltransferases from Citrus paradisi Shivakumar P. Devaiah,†,∥ Daniel K. Owens,§,∥ Mebrahtu B. Sibhatu,†,Δ Tapasree Roy Sarkar,†,Δ Christy L. Strong,†,Δ Venkata K. P. S. Mallampalli,†,Δ Josephat Asiago,† Jennifer Cooke,† Starla Kiser,† Zhangfan Lin,† Anye Wamucho,† Deborah Hayford,† Bruce E. Williams,† Peri Loftis,† Mark Berhow,⊥ Lee M. Pike,† and Cecilia A. McIntosh*,†,‡ †

Department of Biological Sciences, East Tennessee State University, P.O. Box 70703, Johnson City, Tennessee 37614, United States School of Graduate Studies, East Tennessee State University, P.O. Box 70720, Johnson City, Tennessee 37614, United States § Natural Products Utilization Research Unit, ARS, U.S. Department of Agriculture, P.O. Box 1848, University, Mississippi 38677, United States ⊥ Functional Foods Research Unit, ARS, U.S. Department of Agriculture, Peoria, Illinois 61604, United States ‡

S Supporting Information *

ABSTRACT: 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. KEYWORDS: Citrus paradisi, glucosyltransferase, flavonoid, heterologous expression, flavor

1. INTRODUCTION From early investigations into pigmentation roles to extensive recent studies focusing on antioxidant activity and medicinal benefits, flavonoids continue to be an intensively studied class of natural products. They are of specific interest due to their ubiquitous existence in higher plants1−5 and because natural glycosylated flavonoids are the most abundant polyphenols found in plant-based foods.6 In addition to the nutritional aspects of these compounds, they have been of particular focus in Citrus species because they contribute directly to the taste characteristics of fruit and derived products, such as juices. Approximately 60 different flavonoid compounds have been identified in Citrus juices, and many of these compounds contain isomeric diglycosides composed of D-glucose and Lrhamnose.7 It has long been established that the orientation of the linkage between these sugars influences taste in that neohesperidosides (C-1 of rhamnose attached to C-2 of glucose) contribute to astringency and bitterness, whereas rutinosides (C-1 of rhamnose attached to C-6 of glucose) are tasteless.3 This has clear formulation implications for Citrus as neohesperidosides are the predominant components of bitter juices,7,8 whereas rutinosides are more common in sweeter juices. It is also of note, although the compound does not appear to accumulate naturally, that if the bitter flavonoid glycoside neohesperidin is converted to its chalcone form by opening the central ring of the core structure, the resulting © XXXX American Chemical Society

compound is 1500−1800 times sweeter than table sugar at threshold concentrations. This derived synthetic compound (neohesperidin dihydroxychalcone) is used commercially as an artificial sweetener. Grapefruit (Citrus paradisi) is a significant agricultural commodity in the United States, which is routinely second in overall production and first in production for processing (including juicing) worldwide according to recent statistics.9 Aside from grapefruit’s significance as a cash crop, it is also an excellent model system to study the biosynthesis of glycosylated flavonoid derivatives due to its accumulation of a multitude and variety of flavonoid glycosides.3,10−14 Grapefruit is uniquely rich in flavanones and flavones3,10,14 while also producing flavonol 3-O-glucosides, chalcone glycosides, and flavonol 7-O-glycosides in addition to other glycosylated compounds.3,10,12,13 Unlike most other Citrus species, grapefruit accumulates both the tasteless rutinosides and bitter neohesperidosides,3 making it an excellent system in which to examine these commercially important processes. Grapefruit is also known to produce limonin, another natural product that contributes to bitterness. It has been proposed that limonoid Received: November 13, 2015 Revised: February 1, 2016 Accepted: February 18, 2016

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mixture of limonoate and monolactones (enriched in the A-ring monolactone), was lyophilized and stored at −80 °C. 2.2. Plant Material. Seeds of C. paradisi (var. Duncan) were acquired from the Citrus Budwood Registry (Bureau of Citrus Budwood Registration, Winter Haven, FL, USA). Plants were cultivated under outdoor greenhouse conditions (15−24 °C and ambient light). Because prior work has shown that young leaves are the most metabolically active tissues with respect to flavonoid glycosylation,12 RNA was isolated from very young leaves using the RNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA) and Buffer RLC as previously described.32 2.3. Isolation of Putative Glucosyltransferase Genes. Putative glucosyltransferase clones were obtained from young grapefruit leaf using four different approaches as described below and named PGT1− 11.3 Isolation and characterization of PGT1 have been previously described.33 In the first approach, degenerate primers (forward ACNCA(TC)TG(CT)GG*TGGAA(TC)C; reverse GA(AG)TTCCA*CC(GA)CA(AG)TGNGT) were designed against the conserved THCGWN region of the PSPG box motif for use in SMART-RACE (Clontech, Mountain View, CA, USA) RT-PCR to obtain initial partial clones. Subsequently gene-specific primers were designed to primer walk to the coding-region ends to obtain information for full-length coding clones PGT2 and PGT4. Once full-length sequence information was confirmed, gene-specific primers were designed and used in PCR with the leaf cDNA library to obtain full-length clones. PGT5 and PGT6 were identified by the same process, but could not be resolved. In a second approach, a directionally cloned young grapefruit leaf EST cDNA library was constructed, single-pass sequencing from the 5′ end was performed on over 4000 candidates, and results were mined for the presence of a PSPG box.34 PGT3 was obtained through this approach and was full-length upon isolation.34 In a third approach, Cp3GT (previously named PGT7) and PGT8 clones were obtained by analysis of putative Citrus glucosyltransferase sequences posted in the GenBank database at the National Center for Biotechnology Information (NCBI). Primers were designed against an annotated gene from Citrus sinensis (Cp3GT) and another from Citrus unshiu (PGT8), which were subsequently used to amplify full-length coding regions by PCR of the young grapefruit leaf cDNA library. In the last approach, Citrus putative GT contigs derived from partial sequences (predominantly ESTs) across multiple Citrus species were mined from the harvEST bioinformatics database. These contigs were used to design primers that PCR amplified full-length coding-region clones PGT9−11 from the young leaf cDNA library.35 Supplemental Table 1 in the Supporting Information lists the primers used to obtain full-length coding clones of PGTs 2, 3, 4, 7, 8, 9, 10, and 11 as well as to introduce restriction sites for subcloning into expression vectors (see Recombinant Expression). The integrity of all clones was confirmed by restriction digest analysis and by sequencing. 2.4. Phylogenetic Analysis. A multiple alignment of amino acid sequences of C. paradisi flavonol-specific 3-O-glucosyltransferase (GQ141630) along with other C. paradisi putative glucosyltransferases clones was analyzed using BioEdit.36 For phylogenetic analysis, information from functionally characterized flavonoid and limonoid glucosyltransferases was included in an alignment with the amino acid sequences of C. paradisi PGTs, and an unrooted phylogenetic tree was generated using Clustal Omega.37 The evolutionary history was inferred using the neighbor-joining method.38 The bootstrap consensus tree inferred from 500 replicates was taken to represent the evolutionary history of the taxa analyzed. Branches corresponding to partitions reproduced in 98% homology. Clade E contains enzymes that act on a variety of different flavonoid compounds and therefore suggests little in the way of substrate specificity. However, clade E does have implications for regiospecificity as all enzymes in this clade, including Cp3GT, are 3-Oglucosyltransferases. It is also of note that a known flavonol3-O-glucosyltransferase segregates to clade B as well. This indicates that although isolating to clade E is an indication of flavonoid-3-O-glucosyltransferase activity, it does not preclude enzymes from the other clades having the same regioselectivity. Homology-based database searches alone are typically of little use in assigning functional identity of GTs as there are too few sequences in publically available databases with biochemically established function. In a few specific cases, phylogenetic analysis may be of use in narrowing potential GT substrate usage and/or regioselectivity. However, biochemical analysis remains the only reliable method to assign substrate specificity, glycosylation position, and in vivo roles of GTs. New approaches for predictive modeling of GT substrate and

regioselectivity will likely become more robust as the pool of available biochemical characterization data of GTs continues to grow. 3.3. Heterologous Expression and Enrichment of Proteins. All recombinant proteins were initially expressed in a bacterial system. Sufficient soluble protein for biochemical analysis was obtained for PGT2, PGT10, and Cp3GT, although a significant amount of insoluble recombinant protein, which was likely contained within inclusion bodies, was also consistently observed. However, soluble protein yields for the other PGTs were low and/or inconsistent when expressed in E. coli (e.g., Figure 4B), so they were introduced into a yeast recombinant expression system that routinely yielded higher amounts of soluble protein (e.g., Figure 4D). To establish the validity of making comparisons between recombinant proteins produced in the E. coli (pET) and P. pastoris (pPICZ) expression systems, the previously characterized E. coli-expressed Cp3GT was subcloned into pPICZ A. Although the P. pastoris system has C-terminal cMyc/6X His tags as opposed to N-terminal thioredoxin/6X His tags in the E. coli system, Cp3GT showed the same preference for flavonol substrates (Table 2). Similar responses to the presence of metal ions and Ki values for the competitive inhibitor UDP (Table 3), as well as temperature optimum and temperature stability (Figure 5A), were observed.14 The activation energy as determined from an Arrhenius plot was 12.64 kJ mol−1 in P. pastoris, which was similar to the value of 14.4 kJ mol−1 seen in E. coli. Tris buffer had an inhibitory effect on the activity of Cp3GT in both P. pastoris and E. coli, which further confirmed that this previously observed result was not an artifact resulting from recombinant expression in a prokaryotic system (Figure 5B). The primary difference between Cp3GT in the two systems was that when the enzyme was expressed in P. pastoris, it had increased stability at higher pH values.14 This could be due to the chemistry and/or placement of tags as others using both systems have seen similar increase in pH stability/activity with enzymes produced in the P. pastoris system with identical tags (Kumar, personal communication). G

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Journal of Agricultural and Food Chemistry Table 2. Glucosyltransferase Activity of C. paradisi PGT Clones with Flavonoid Substrates

a

Data are relative percent incorporation of 14C-labeled UDP-glucose as compared to Cp3GT using quercetin as substrate (n = 2).

3.4. Functional Characterization and Substrate Specificity. After expression of proteins in E. coli and/or P. pastoris, purified proteins were tested for GT activity with representative flavonoid, coumarin (umbelliferone, esculetin, scopotelin, ocoumaric acid), and/or simple phenolic substrates (2,4dihydrobenzaldehyde, catechol, salicylic acid, 3,4-dihydrobenzoic acid, sinapinic acid, caffeic acid, phloroacetophenone, phydroxyphenylacetic acid, p-hydroxyphenylpyruvate, gentisic

acid, vanillic acid, vanillin), which are predominantly tri-, di-, and monocyclic phenolics, respectively. Table 2 summarizes results obtained with flavonoid substrates. GT activity was not detected toward any of the flavonoid, coumarin, or simple phenolic substrates tested with empty pCD1 and pPICZ A vector negative controls or with PGT4, PGT9, PGT10, and PGT11 (Table 2).44,47,48 H

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glycosylation.50 These results also further underscore that, at this time, primary sequence analysis alone is typically not a reliable tool for functional assignment as homology-based searches resulted in incorrect predictions for each of the above enzymes. No detectable activity for PGT2 with coumarins or simple phenolics was observed with the conditions used. However, a low level of GT activity with quercetin as an acceptor substrate was detected. The amount of reaction product was insufficient for the detection by HPLC analysis, but the radiolabeled product did cochromatograph with quercetin-3-O-glucoside by TLC (Supplemental Figure 1), suggesting that its activity mirrored that of Cp3GT. However, in contrast, the activity was only 26% of what was observed with Cp3GT, and no measurable activity was seen with any of the other flavonols tested (Table 2). PGT2 and Cp3GT have unique variations in their expression patterns.32 These differences in expression along with variability in enzymatic activity involving flavonols could have an impact upon the nature of the flavonoid compounds that accumulate in various C. paradisi tissues. However, it has been observed in a number of cases that if GTs are promiscuous in regard to substrate, they will often show some level of activity with quercetin. This may be due in part to flavonol-3-O-GTs representing a parental group from which the other GT classes have evolved, as has been suggested by phylogenetic analysis. Therefore, the possibility that PGT2 has activity with substrates beyond what has been seen at this time cannot be excluded. Significant activity with flavonoids (Table 2) or coumarins was not detected with PGT3. However, HPLC analysis of enzyme screening reactions using simple phenolics as acceptor substrates did indicate GT activity with the simple phenolic compound, catechol (Figure 6), and we report this initial result here. Catechol is an agriculturally important compound having a well-known role in browning (e.g., apple and potato), as well as commonly being used as a base structure in the production of synthetic pesticides. Because our focus was on characterizing flavonoid glucosyltransferases, further kinetic analyses were not performed on the PGT3 protein. Isotope-based assays were not possible with this substrate as its structure precludes the EtOAc liquid−liquid extraction necessary for the separation of

Table 3. Effect of Metals and UDP on Activity of Cp3GT Expressed in E. coli and in P. pastoris Cp3GT expressed in E. coli, % relative activitya,b

Cp3GT expressed in P. pastoris, % relative activitya

compound

10 mM

1 mM

10 mM

1 mM

untreated UDPc CaCl2 KCl NaCl ZnCl2 FeSO4 CuSO4

100 6 94 97 95 26 0.9 0.03

100 25 104 102 102 29 0.78 0.08

100 1 97 97 95 24 0.6 1

100 7 98 97 96 28 1 1

a

Data are results using quercetin as acceptor. bResults from Owens and McIntosh.14 cKi,app (competitive inhibition) for UDP was 69.5 μM for E. coli expressed protein with N-terminal thioredoxin/6X His tags and 87.1 μM for P. pastoris expressed protein with C-terminal Myc/6X His tags.

PGT8 coded for a protein with 98% identity to the limonoid GT enzyme inferred from the C. unshiu gene used for its initial identification. Enzymatic activity has been demonstrated for the C. unshiu enzyme; however, it was not extremely active, requiring reactions to be performed for 3 h to produce detectable amounts of product.42 We replicated as well as modified, extending assay time well beyond 3 h, these reaction conditions for limonoid GT activity. No clear limonoid GT activity was detected with any of the conditions used. Subsequently, PGT8 was tested with a suite of flavonoids, partially motivated by the fact that some terpenoid GTs have previously been shown to use quercetin as a substrate.49 No enzyme activity was observed with flavonoid substrates. Whereas the identification of a preferred substrate provides more definitive information with regard to GT structure/ function, eliminating potential substrates is also a critical step in refining GT function. These results are in line with previous observations from the literature. For example, of the 107−120 PGTs that have been suggested to occur in the Arabidopsis genome, only 7 have been shown to be involved in flavonoid

Figure 5. Characterization of grapefruit flavonol 3-O-glucosyltransferase, Cp3GT, expressed in P. pastoris: (A) effect of temperature on enzyme activity (TE, reaction incubation temperature; TS, temperature sensitivity/pre-incubation temperature); (B) pH profile with activity levels measured in MES (○), phosphate (△), bicine (□), CHES (◇), or Tris (●). Activity was measured using quercetin as acceptor; n = 2. I

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Figure 6. PGT3 enzyme assay with catechol as substrate and detection via HPLC. (A) Catechol standard. The retention time of the standard peak is 2.799 min (λ = 280). (B) GT reaction. Note the decrease in area of the aglycone peak and appearance of a peak at an earlier retention time (1.409). As catechol is a symmetrical molecule, whichever hydroxyl group is modified will result in the production of an equivalent structure. There is not sufficient separation between substrate and product to suggest the synthesis of a diglycoside.

Figure 7. HPLC chromatograms for GT assay conducted with crude grapefruit leaf extract with and without added PGT9-encoded protein: (A) quercetin standard; (B) GT reaction with crude grapefruit leaf extract (positive control); (C) GT reaction with crude grapefruit leaf extract and added PGT9-encoded protein. Arrows indicates potential glucosylated product in HPLC chromatogram.

unincorporated radiolabeled glucose from the reaction product. The catechol molecule contains two hydroxyl groups that could serve as potential sites of glucosylation (Figure 6). Although the specific glucosylation site was not determined in our studies, the symmetrical nature of the molecule would result in the formation of the same catechol-O-glucoside after incorporation of glucose at either position. The retention times observed

between the aglycone and product were consistent with the synthesis of a monoglucoside. Although PGT9 had no discernible enzyme activity with any of the substrates tested under the assay conditions used, evidence of activity was observed. As part of the PGT assay screening process, assays including quercetin as substrate and crude C. paradisi young leaf extract as the enzyme source are routinely included as a positive control because these reactions J

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result in easily detectable amounts of radiolabeled glucosides. Separate reactions containing a recombinantly expressed PGT in the reaction mixture with the positive control described above are also performed to establish if a recombinant enzyme may have any inhibitory action on GT reactions. Inhibitory activity was never observed with any of the PGTs; however, when this control was performed with PGT9, a significant increase in radioactive signal was observed. Subsequent HPLC analysis of the reaction performed with unlabeled UDP-glucose resulted in the disappearance of one peak and an increase in the size of an earlier eluting peak as compared to the assay performed with crude grapefruit young leaf extract alone (Figure 7). These results are consistent with the formation of a glucosylated product. This might indicate the presence of an unscreened native substrate in the crude extract at high enough levels to result in detectable enzyme activity or, intriguingly, could suggest the presence of other protein(s) necessary to complement enzyme activity, such as those associated in a flavonoid metabolon. Further analyses with PGT9 and investigations into these findings are planned. This work is a culmination of the efforts of undergraduate, graduate, and postdoctoral scholars to identify and elucidate the biochemical function of PGTs from C. paradisi. These efforts have resulted in the identification of nine resolved PGT sequences to date. Of the identified PGTs, flavonol 3-O-GT activity has been thoroughly established for Cp3GT,14 quercetin 3-O-GT activity has been identified for PGT2, catechol GT activity has been shown for PGT3, and an as yet unresolved GT activity has been observed with PGT9. These results are an important step in further understanding the functioning of GTs in general and of grapefruit in particular. As such, it is also a critical contribution toward achieving the future design of custom GT enzymes for the production of desired compounds.

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This work was supported by USDA Grant 2003-35318-13749 awarded to C.A.M. and L.M.P.; NSF MCB Grants 0614260 and 1120268 and NSF MCB REU supplements 0734216, 0829613, 1228176, and 1418501 awarded to C.A.M.; and a Sigma Xi GIAR awarded to T.R.S. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge Brenda Winkel of Virginia Tech for the suggestion of pCD1 and Sanja Roje of Washington State University for the recommendation of the pPICZ A as well as L. Epling, P. Campbell, and J. Cantrell for expert technical assistance.

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ABBREVIATIONS USED EST, expressed sequence tag; GT, glycosyltransferase; IMAC, immobilized metal affinity chromatography; IPTG, isopropyl-βD-1-thiogalactopyranoside; PGT, putative glucosyltransferase; PSPG, plant secondary product glycosyltransferase; Trx, thioredoxin; UDP, uridine diphosphate; UGT, uridine diphosphate activated glycosyltransferase

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b05430. Supplemental Table 1, primers used to obtain full-length coding regions from the cDNA library and introduce restrictions cites for subcloning; Supplemental Table 2, a percentage identity matrix among the identified CpPGTs; Supplemental Figure 1, identification of the PGT2 reaction product (PDF)

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AUTHOR INFORMATION

Corresponding Author

*(C.A.M.) Phone: (423) 439-6147. Fax: (423) 439-5958. Email: [email protected]. Present Address Δ

(M.B.S.) ImClone Systems, 450 E. 29th St., New York, NY 10016, USA. (T.R.S.) MD Anderson Cancer Center, The University of Texas, 1515 Holcombe Blvd., Houston, TX 77030, USA. (C.L.S.) School of Life Sciences, University of NevadaLas Vegas, Box 4004, 4505 S. Maryland Parkway, Las Vegas, NV 89154, USA. (V.K.P.S.M.) The University of Texas Health Science Center at Houston, 7000 Fannin, Suite 1200, Houston, TX 77030, USA. Author Contributions ∥

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DOI: 10.1021/acs.jafc.5b05430 J. Agric. Food Chem. XXXX, XXX, XXX−XXX