Novel Insights Into the Existence of the Putative UDP-Glucuronate 5-Epimerase Speciﬁcity

catalysts

Article Novel Insights into the Existence of the Putative UDP-Glucuronate 5-Epimerase Speciﬁcity

Ophelia Gevaert, Stevie Van Overtveldt, Matthieu Da Costa, Koen Beerens and Tom Desmet *

Centre for Synthetic Biology, Department of Biotechnology, Ghent University, Coupure Links 653, 9000 Gent, Belgium; [email protected] (O.G.); [email protected] (S.V.O.); [email protected] (M.D.C.); [email protected] (K.B.) * Correspondence: [email protected]; Tel.: +32-9264-9920

Received: 15 January 2020; Accepted: 9 February 2020; Published: 12 February 2020

Abstract: C5-epimerases are promising tools for the production of rare l-hexoses from their more common d-counterparts. On that account, UDP-glucuronate 5-epimerase (UGA5E) attracts attention as this enzyme could prove to be useful for the synthesis of UDP-l-iduronate. Interestingly, l-iduronate is known as a precursor for the production of heparin, an effective anticoagulant. To date, the UGA5E specificity has only been detected in rabbit skin extract, and the respective enzyme has not been characterized in detail or even identified at the molecular level. Accordingly, the current work aimed to shed more light on the properties of UGA5E. Therefore, the pool of putative UGA5Es present in the UniProt database was scrutinized and their sequences were clustered in a phylogenetic tree. However, the examination of two of these enzymes revealed that they actually epimerize UDP-glucuronate at the 4- rather than 5-position. Furthermore, in silico analysis indicated that this should be the case for all sequences that are currently annotated as UGA5E and, hence, that such activity has not yet been discovered in nature. The detected l-iduronate synthesis in rabbit skin extract can probably be assigned to the enzyme chondroitin-glucuronate C5-epimerase, which catalyzes the conversion of d-glucuronate to l-iduronate on a polysaccharide level.

Keywords: UDP-glucuronate 5-epimerase; UDP-glucuronate 4-epimerase; CEP1 family; l-iduronate; biocatalysis; phylogenetic tree; recombinant expression

1. Introduction Carbohydrate epimerases (CEP, EC 5.1.3) catalyze the inversion of the configuration of a chiral carbon atom in molecules with more than one stereocenter [1]. Accordingly, they are able to create shortcuts in the production of rare sugars starting from their abundant counterparts [2]. Over 30 different carbohydrate epimerases have been described to date, covering various substrate and product specificities. Despite their seemingly simple net reaction, the mechanisms employed by these enzymes are diverse and sometimes quite complex. Recently, a comprehensive classification was proposed for epimerases, which consists of 14 families (CEP1–CEP14) in which structure and mechanism are conserved [3]. Thereof, the CEP1 family is the largest, containing 12 different specificities on nucleoside diphosphate (NDP) sugars that share a mechanism based on a transient keto intermediate (Table1). This family is a rich pool of epimerases displaying diverse substrate and product specificities. Interestingly, this means that comparable active site architectures are able to bind different substrate molecules and yield varying reaction products.

Catalysts 2020, 10, 222; doi:10.3390/catal10020222 www.mdpi.com/journal/catalysts Catalysts 2020, 10, 222 2 of 11

Table 1. Overview of carbohydrate epimerase family 1. The speciﬁcities marked with an asterisk have a solved crystal structure.

EC Number Speciﬁcity 5.1.3.2 UDP-glucose 4-epimerase * 5.1.3.10 CDP-paratose 2-epimerase * 5.1.3.6 UDP-glucuronate 4-epimerase * 5.1.3.7 UDP-N-acetylglucosamine 4-epimerase * 5.1.3.16 UDP-glucosamine 4-epimerase 5.1.3.25 dTDP-l-rhamnose 4-epimerase 5.1.3.26 N-acetyl-α-d-glucosaminyl-diphospho-ditrans, octacis-undecaprenol 4-epimerase 5.1.3.5 UDP-l-arabinose 4-epimerase 5.1.3.12 UDP-glucuronate 5-epimerase 5.1.3.20 ADP-l-glyceromanno-heptose 6-epimerase * / UDP-2-acetamido-2,6-dideoxy-beta-l-arabino-4-hexulose 3-epimerase 5.1.3.18 GDP-mannose 3,5-epimerase *

Based on the CEP classification, two C5-epimerases are present in the CEP1 family. These enzymes are of interest as they might contribute to the production of l-hexoses and derivatives, with applications as for instance antibiotics or nucleosides [4–6]. On the one hand, the double epimerization of GDP-mannose yielding GDP-l-galactose and GDP-l-gulose is catalyzed by GDP-mannose 3,5-epimerase (GM35E, EC 5.1.3.18), an enzyme that has already been thoroughly characterized [7,8]. On the other hand, the UDP-glucuronate 5-epimerase specificity (UGA5E, EC 5.1.3.12) would ensure the interconversion of UDP-d-glucuronic acid (UDP-GlcA) and UDP-l-iduronic acid (UDP-IdoA) (Figure1)[ 9]. This enzyme might be industrially relevant as UDP-IdoA is known as a precursor of heparin, a glycosaminoglycan (GAG) consisting of sulfated disaccharide repeating units of uronic acids (GlcA or IdoA) and d-glucosamine residues. This compound displays anticoagulant properties and is already practiced clinically [10,11]. Currently, the drug is isolated from animal tissue, yielding a heterogenous mixture [12]. However, research is focusing on the establishment of a (bio)chemical production process. One of the hurdles there is the synthetic access to l-iduronic acid and l-idose as building blocks [13]. In that respect, UGA5E might prove to be a very useful biocatalyst. In addition, the structural characterization of UGA5E might contribute to a better understanding of the CEP1 specificity determinants. Indeed, the comparison of the reaction mechanisms of GM35E and UGA5E could give more insight into the specific parameters that potentially promote or prevent a double epimerization,Catalysts 2020, 10, respectively. 222 3 of 11

Figure 1. Epimerization of UDP-glucuronic acid (UDP-GlcA) toward UDP-l-iduronic acid (UDP-IdoA)Figure 1. Epimerization and UDP-galacturonic of UDP-glucuronic acid (UDP-GalA) acid (UDP-GlcA) by UDP-glucuronate toward UDP-L-iduronic 5-epimerase acid (UGA5E)(UDP- andIdoA) UDP-glucuronate and UDP-galacturonic 4-epimerase acid (UDP-GalA) (UGA4E), respectively. by UDP-glucuronate 5-epimerase (UGA5E) and UDP- glucuronate 4-epimerase (UGA4E), respectively. To date, UGA5E activity has only been detected in rabbit skin extracts, where the enzyme was believed2. Results to play a role in the synthesis of dermatan sulfate (DS, formerly chondroitin sulfate B) [9]. The structure of this GAG is comparable to heparin, but DS is composed of N-acetyl-d-galactosamine 2.1. Selection of a UGA5E Sequence (GalNAc) building blocks instead of d-glucosamine residues [14]. Moreover, the IdoA subunits even functionThe UniProt as key database components contains in 1154 the bindingputative site‘UDP-glucuronate speciﬁcity for 5-epimerase’ GAG-binding sequences, proteins of [15 ]. Thewhich compound 20 are isderived expressed from in eukaryotes, various mammalian 10 from archaea, tissues and and the is others involved from in bacteria. cardiovascular One is disease,even tumorigenesis,listed as a reviewed infection, sequence, wound but repair, this enzyme and ﬁbrosis. from Rhizobium Jacobson meliloti et al. proposed (RmUGAE, a three-stageUniProt: O54067) process is most likely incorrectly annotated as the literature accompanying this entry actually discusses an for the production of this polysaccharide, starting from the conversion of UDP-glucose (UDP-Glc) UGA4E [21]. Consequently, this sequence was excluded from further analysis. As the UGA5E specificity might be valuable in an industrial context, a (thermo)stable representative from a bacterial source would be most relevant. Indeed, a bacterial variant should enable facilitated recombinant expression in prokaryotes, and (thermo)stability entails a higher tolerance for the harsh process conditions practiced in industry [22,23]. Therefore, the UniProt database was screened for UGA5E enzymes that meet these conditions. This analysis highlighted three promising sequences, namely, those from Thermacetogenium phaeum (UniProt: K4LEK1), Desulfurobacterium thermolithotrophum strain DSM 11699 (UniProt: F0S202), and Thermodesulfobacterium geofontis (UniProt: F8C4X8). The optimal growth temperatures of these organisms are 58, 70, and 83 °C, respectively [24–27]. For the current study, the highest temperature was selected, i.e., the putative UGA5E from T. geofontis (TgUGAE).

2.2. Evaluation of TgUGAE First, the inducible expression of TgUGAE in E. coli was evaluated and seemed to be the highest at an incubation temperature of 20 °C (Figure S1). As it was found that the enzyme was mainly expressed in the insoluble fraction, activity tests were performed with crude cell lysate. The conversion of UDP-GlcA was followed over time and the formation of one reaction product was observed. The retention time of the hydrolyzed product on HPLC corresponded to that of GalA, the C4-epimer of GlcA (Figure 2). Further analysis confirmed that this detected UGA4E activity was derived from the overexpressed TgUGAE sequence and not from the accompanying E. coli background (Figure S2). The possibility that the biocatalyst would display a side activity as C5- epimerase was disproved as no extra product was obtained, even after extensive incubation and spiking the reaction with fresh lysate. Furthermore, activity tests with purified enzyme and 5 mM additional NAD+ resulted in the same reaction profile. The extra peak in the chromatogram at 5.70 min was also present in control samples and was, thus, not the result of any enzymatic activity (Figure S3). Consequently, it could be concluded that the putative UGA5E sequence from Thermodesulfobacterium geofontis is actually an UGA4E, with a specific activity of 1.7 (± 0.2) U/mg (using 10 mM UDP-GlcA at pH 7.5 and 30 °C).

Catalysts 2020, 10, 222 3 of 11 to UDP-GlcA by the action of an UDP-glucose dehydrogenase. Next, UGA5E would catalyze the synthesis of UDP-IdoA from UDP-GlcA. Eventually, l-iduronic acid would be transferred to DS by a glycosyltransferase [9]. This pathway is comparable to that involving UDP-glucuronate 4-epimerase (UGA4E, EC 5.1.3.6), an enzyme that catalyzes the interconversion of UDP-GlcA and UDP-d-galacturonic acid (UDP-GalA) (Figure1). The latter serves as a donor for GalA incorporation in plant cell walls, bacterial lipopolysaccharides, or antibiotics [16–20]. UGA4E is also present in the CEP1 family [3]. Despite the detection of UGA5E activity in the tissue extracts, the respective gene was never identiﬁed or recombinantly expressed. Nevertheless, sequence databases contain various putative UGA5Es on which their assignment to the CEP1 family is based. On that account, this research focused on the analysis of these putative sequences and on the UGA5E speciﬁcity in general.

2. Results

2.1. Selection of a UGA5E Sequence The UniProt database contains 1154 putative ‘UDP-glucuronate 5-epimerase’ sequences, of which 20 are derived from eukaryotes, 10 from archaea, and the others from bacteria. One is even listed as a reviewed sequence, but this enzyme from Rhizobium meliloti (RmUGAE, UniProt: O54067) is most likely incorrectly annotated as the literature accompanying this entry actually discusses an UGA4E [21]. Consequently, this sequence was excluded from further analysis. As the UGA5E speciﬁcity might be valuable in an industrial context, a (thermo)stable representative from a bacterial source would be most relevant. Indeed, a bacterial variant should enable facilitated recombinant expression in prokaryotes, and (thermo)stability entails a higher tolerance for the harsh process conditions practiced in industry [22,23]. Therefore, the UniProt database was screened for UGA5E enzymes that meet these conditions. This analysis highlighted three promising sequences, namely, those from Thermacetogenium phaeum (UniProt: K4LEK1), Desulfurobacterium thermolithotrophum strain DSM 11699 (UniProt: F0S202), and Thermodesulfobacterium geofontis (UniProt: F8C4X8). The optimal growth temperatures of these organisms are 58, 70, and 83 ◦C, respectively [24–27]. For the current study, the highest temperature was selected, i.e., the putative UGA5E from T. geofontis (TgUGAE).

2.2. Evaluation of TgUGAE First, the inducible expression of TgUGAE in E. coli was evaluated and seemed to be the highest at an incubation temperature of 20 ◦C (Figure S1). As it was found that the enzyme was mainly expressed in the insoluble fraction, activity tests were performed with crude cell lysate. The conversion of UDP-GlcA was followed over time and the formation of one reaction product was observed. The retention time of the hydrolyzed product on HPLC corresponded to that of GalA, the C4-epimer of GlcA (Figure2). Further analysis confirmed that this detected UGA4E activity was derived from the overexpressed TgUGAE sequence and not from the accompanying E. coli background (Figure S2). The possibility that the biocatalyst would display a side activity as C5-epimerase was disproved as no extra product was obtained, even after extensive incubation and spiking the reaction with fresh lysate. Furthermore, activity tests with purified enzyme and 5 mM additional NAD+ resulted in the same reaction profile. The extra peak in the chromatogram at 5.70 min was also present in control samples and was, thus, not the result of any enzymatic activity (Figure S3). Consequently, it could be concluded that the putative UGA5E sequence from Thermodesulfobacterium geofontis is actually an UGA4E, with a specific activity of 1.7 ( 0.2) U/mg (using 10 mM UDP-GlcA at pH 7.5 and 30 C). ± ◦ Catalysts 2020, 10, 222 4 of 11 Catalysts 2020, 10, 222 4 of 11

FigureFigure 2. Product 2. Product formation formation by Tg byUGAE TgUGAE crude crude cell lysate. cell lysate. (a) Chromatographic (a) Chromatographic analysis analysis of the reactionof the mixturereaction (blue). mixture The peaks(blue). were The comparedpeaks were to compared known standards to known (black). standards The (black). nucleoside The diphosphatenucleoside (NDP)-sugarsdiphosphate of the(NDP)-sugars reaction mixture of the were reaction hydrolyzed mixture to theirwere respective hydrolyzed monosaccharides to their respective prior to analysis.monosaccharides GalA: galacturonic prior to analysis. acid; GlcA: GalA: glucuronic galacturonic acid; acid; IdoA: GlcA:l -iduronicglucuronic acid. acid; (IdoA:b) Conversion L-iduronic of UDP-glucuronicacid. (b) Conversion acid toward of UDP-glucuronic UDP-galacturonic acid toward acid over UDP-galacturonic time. acid over time.

2.3.2.3. In Silico In Silico Analysis Analysis of theof the UGA5E UGA5E Specificity Specificity TheThe further further quest quest for for a truea true UGA5E UGA5E was was assisted assisted byby anan inin silico approach.approach. Commonly, Commonly, unknown unknown genesgenes are are annotated annotated based based on on sequence sequence similarities. similarities. AsAs nono reference sequence sequence is is available available for for UGA5E, UGA5E, the annotationthe annotation of potential of potential UGA5E UGA5E sequences sequences is rather is arbitrary.rather arbitrary. The hypothesized The hypothesized UGA5E specificity,UGA5E andspecificity, the CEP1 family,and the in CEP1 general, family, is partin general, of the is short-chain part of the dehydrogenaseshort-chain dehydrogenase/reductase/reductase (SDR) superfamily, (SDR) a largesuperfamily, cluster of a large NAD(P)(H)-dependent cluster of NAD(P)(H)-dependent oxidoreductases oxidoreductases [3,28]. Members [3,28]. Members of this familyof this family display lowdisplay sequence low identitiessequence identities (typically (typically 15–30%), 15–30%), though though they sharethey share some some common common motifs motifs such such as as an + GxxGxxGan GxxGxxG motif formotif NAD(P) for NAD(P)+ binding binding within within a Rossmann a Rossmann fold fold and and an an YxxxK YxxxK catalytic catalytic motif. motif. Based Based on theseon features, these features, sequences sequences can be classifiedcan be classified in the SDR in superfamily.the SDR superfamily. Subsequently, Subsequently, specific conservedspecific conserved residues and secondary structures can contribute to the distinction of various enzymatic residues and secondary structures can contribute to the distinction of various enzymatic specificities, specificities, e.g., the conserved catalytic cysteine and lysine in GM35E [8]. Due to the lack of a e.g., the conserved catalytic cysteine and lysine in GM35E [8]. Due to the lack of a reference sequence reference sequence for UGA5E, the determination of these specific conserved residues is thus for UGA5E, the determination of these specific conserved residues is thus unachievable at the moment. unachievable at the moment. On that account, the actual function of the putative UGA5E sequences On thatis dubious. account, the actual function of the putative UGA5E sequences is dubious. ThisThis statement statement is clearlyis clearly demonstrated demonstrated when when performingperforming a Basic Local Local Alignment Alignment Search Search Tool Tool (BLAST)(BLAST) analysis analysis with with the theTgUGAE TgUGAE sequence. sequence. Indeed, Indeed, 19% 19% of of the the resulting resulting sequences sequences are are annotated annotated as ‘NAD-dependentas ‘NAD-dependent epimerase epimerase/dehydratase/oxidoreduc/dehydratase/oxidoreductase,’tase,’ which which implies implies that that they they are are part part of theof the SDR superfamilySDR superfamily and this and annotation this annotation is, thus, is, most thus, probably most probably based onbased the on sequence the sequence motifs. motifs. Another Another 72% of the BLAST72% of the outcome BLAST consists outcome of consists ‘NDP-sugar of ‘NDP-sugar epimerase’ epimerase’ annotated annotated sequences. sequences. The remaining The remaining results (9%)results comprise (9%) a comprise variety ofa variety specific of epimerases, specific epimerases, such as UGA5Essuch as UGA5Es and UGA4Es. and UGA4Es. In orderIn order to findto find a sequence a sequence with with a a high high probability probability ofof beingbeing an UGA5E, UGA5E, a a phylogenetic phylogenetic tree tree of ofall all putativeputative UGA5E UGA5E sequences sequences was was constructed constructed (Figure (Figure3). Next,3). Next, ten ten bacterial bacterial sequences sequences from from around around the treethe were tree randomly were randomly selected selected for further for further investigation. investigation. A BLAST A BLAST search search was performedwas performed with with each each of the sequencesof the sequences and the ratio and of the ‘UDP-glucuronate ratio of ‘UDP-glucuronate 5-epimerase’ 5-epimerase’ as the outcome as the comparedoutcome compared to all results to all was analyzedresults (Figure was analyzed4). It was (Figure hypothesized 4). It was that hypothesized enzymes with that a enzymes high ratio with have a ahigh bigger ratio chance have toa bigger display chance to display the UGA5E specificity as the annotation is based on a common feature among the the UGA5E specificity as the annotation is based on a common feature among the putative UGA5E putative UGA5E sequences. Accordingly, the UGA5E from Agrobacterium radiobacter strain K84 sequences. Accordingly, the UGA5E from Agrobacterium radiobacter strain K84 (ArUGAE, UniProt: (ArUGAE, UniProt: B9J8R3) was selected as being the sequence with the highest ratio (59%). B9J8R3) was selected as being the sequence with the highest ratio (59%). Interestingly, according to this Interestingly, according to this analysis, TgUGAE displayed a very low ratio of only 3%. The fact that analysis, TgUGAE displayed a very low ratio of only 3%. The fact that this enzyme was then actually a this enzyme was then actually a C4-epimerase substantiates the above reasoning. C4-epimerase substantiates the above reasoning.

Catalysts 2020, 10, 222 5 of 11

CatalystsCatalysts2020, 102020, 222, 10, 222 5 5of of 11 11

Figure 3. Phylogenetic tree of the putative UGA5E sequences present in the UniProt database. Eukaryotic sequences are colored in green, whereas the sequences derived from archaea are Figurehighlighted 3. Phylogenetic in blue. A tree BLAST of the search putative was performed UGA5E sequences with the sequences present in represented the UniProt in database.orange. EukaryoticTgFigureUGAE sequences 3. is Phylogeneticpresented are coloredin red.tree The inof green,the arrows putative whereas indicate UGA5E the th sequencese sequences sequences derived that present were from inexperimentally archaeathe UniProt are highlighted testeddatabase. in in blue.thisEukaryotic research. A BLAST sequences search wasare colored performed in green, with thewhereas sequences the sequences represented derived in orange. from archaeaTgUGAE are is presentedhighlighted in red. in The blue. arrows A BLAST indicate search the sequenceswas performed that were with experimentally the sequences testedrepresented in this in research. orange. TgUGAE is presented in red. The arrows indicate the sequences that were experimentally tested in this research.

Figure 4. Ratio of ‘UDP-glucuronate 5-epimerase’ sequences in the BLAST results of the randomly selectedFigure putative 4. Ratio UGA5E of ‘UDP-glucuronate sequences. The 5-epimerase’ sequences are sequences represented in the by BLAST their UniProt results of entry. the randomlyTgUGAE is indicatedselected by putative an arrow. UGA5E sequences. The sequences are represented by their UniProt entry. TgUGAE is indicated by an arrow. 2.4. EvaluationFigure of 4. ArUGAE Ratio of ‘UDP-glucuronate 5-epimerase’ sequences in the BLAST results of the randomly 2.4.The Evaluation recombinantselected putative of ArUGAE expression UGA5E sequences. of ArUGAE The was,sequen again,ces are first represented evaluated, by their and UniProt an incubation entry. Tg temperatureUGAE is indicated by an arrow. of 20 ◦CThe was recombinant found to be mostexpression suitable of (FigureArUGAE S4). was, As wasagain, the first case evaluated, for TgUGAE, and thean enzymeincubation was mainlytemperature present inof the20 °C insoluble was found fraction to be and,mostthus, suitable the (Figure activity S4). tests As were was performedthe case for withTgUGAE, crude the cell 2.4. Evaluation of ArUGAE lysate.enzyme As can was be mainly seen inpresent Figure in5 the, this insoluble enzyme fraction also catalyzes and, thus, the the interconversion activity tests were of performed UDP-GlcA with and UDP-GalAcrude Thecell and lysate.recombinant thus As displays can beexpression seen an UGA4E in Figure of specificityAr 5,UGAE this enzyme was, instead again,also of catalyzes thefirst hypothesized evaluated, the interconversion and UGA5E an incubation specificity.of UDP- Again,GlcAtemperature extra and controls UDP-GalA of 20 were °C and was included thus found displays toto eliminatebe mostan UGA4E suitable the possibility specificity (Figure S4). thatinstead As the was of UGA4E the the hypothesized case activity for Tg wasUGAE, UGA5E derived the enzyme was mainly present in the insoluble fraction and, thus, the activity tests were performed with fromspecificity. the E. coli Again,background extra controls and to were assign included the extra to eliminate peak in thethe chromatogrampossibility that the (Figures UGA4E S5 activity and S6). wascrude derived cell lysate. from theAs canE. coli be backgroundseen in Figure and 5, tothis assign enzyme the alsoextra catalyzes peak in thethe chromatograminterconversion (Figures of UDP- Moreover, the addition of NAD+ had no influence on the epimerization specificity. Accordingly, GlcA and UDP-GalA and thus displays an UGA4E specificity instead of the hypothesized UGA5E this sequence from Agrobacterium radiobacter should be designated as UGA4E. The enzyme displayed a specificity. Again, extra controls were included to eliminate the possibility that the UGA4E activity specific activity of 12.6 ( 1.2) U/mg (using 10 mM UDP-GlcA at pH 7.5 and 30 C). was derived from the± E. coli background and to assign the extra peak in the chromatogram◦ (Figures

Catalysts 2020, 10, 222 6 of 11

S5 and S6). Moreover, the addition of NAD+ had no influence on the epimerization specificity. CatalystsAccordingly,2020, 10, 222 this sequence from Agrobacterium radiobacter should be designated as UGA4E. 6The of 11 enzyme displayed a specific activity of 12.6 (± 1.2) U/mg (using 10 mM UDP-GlcA at pH 7.5 and 30 °C).

FigureFigure 5. Specificity 5. Specificity testing testing of ArUGAE of ArUGAE crude cellcrude lysate. cell (lysate.a) Product (a) Product analysis analysis on HPLC on of HPLC the Ar UGAEof the reactionArUGAE mixture reaction (blue). mixture Standards (blue). are Standards represented are inre black.presented The in reaction black. The mixture reaction was mixture hydrolyzed was priorhydrolyzed to analysis toprior obtain to freeanalysis monosaccharides. to obtain free GalA: monosaccharides. galacturonic acid; GalA: GlcA: galacturonic glucuronic acid; acid; GlcA: IdoA: l-iduronicglucuronic acid. acid; (b) Conversion IdoA: L-iduronic of UDP-glucuronic acid. (b) Conversion acid to of UDP-galacturonic UDP-glucuronic acid acid to over UDP-galacturonic time. acid over time. 2.5. Analysis of the UGA5E Specificity 2.5. Analysis of the UGA5E Specificity Both examined putative UGA5E sequences actually appeared to be UGA4Es. These findings raise questionsBoth about examined the specificity putative of theUGA5E other sequences sequences actua in thelly phylogeneticappeared to be tree. UGA4Es.ArUGAE These was findings selected basedraise on its questions high UGA5E about ratiothe specificity among the of BLAST the other results. sequences However, in the it phylogenetic is very likely tree. that Ar theUGAE presumed was UGA5Eselected from basedRhizobium on its melilotihigh UGA5E, which ratio actually among is the an UGA4E,BLAST results. was used However, as a reference it is very for likely annotation. that the Consequently,presumed this UGA5E would from mean Rhizobium that UGA4E-specific meliloti, which featuresactually areis an assigned UGA4E, to was the used hypothesized as a reference UGA5E for specificity.annotation. Accordingly, Consequently, chances this were would high that meanAr UGAEthat UGA4E-specific displayed the UGA4Efeatures specificity. are assigned In addition, to the hypothesized UGA5E specificity. Accordingly, chances were high that ArUGAE displayed the detailed analysis of the BLAST results of ArUGAE showed that RmUGAE was one of the outcomes. UGA4E specificity. In addition, detailed analysis of the BLAST results of ArUGAE showed that When a reverse reasoning is applied, this would mean that sequences with a low UGA5E ratio are RmUGAE was one of the outcomes. When a reverse reasoning is applied, this would mean that more likely to actually display UGA5E specificity. Nevertheless, even though the ratio of TgUGAE sequences with a low UGA5E ratio are more likely to actually display UGA5E specificity. was indeedNevertheless, low, the even enzyme though still the displayedratio of TgUGAE UGA4E was activity. indeed Takinglow, the into enzyme account still displayed the fact that UGA4E both epimerasesactivity. are Taking located into in diaccountfferent the clades fact ofthat the both phylogenetic epimerases tree, are it located is hypothesized in different that clades the UGA4E of the specificityphylogenetic can be assignedtree, it is to hypothesized all of these sequences. that the UGA4E specificity can be assigned to all of these Thissequences. hypothesis is supported by a more detailed sequence analysis. Sun et al. recently solved the first crystalThis structure hypothesis of anis supported UGA4E (i.e., by a frommore Streptomycesdetailed sequence viridosporus analysis., PDBSun et 6KV9) al. recently and foundsolved thatthe crucialfirst interactions crystal structure are formed of an UGA4E with the (i.e., substrate from Streptomyces at positions viridosporus G92 and R192, PDB [ 206KV9)]. Moreover, and found R192 that is believedcrucial tofavor interactions the sugar are ringformed rotation with thatthe substrate is needed at for positions C4-epimerization, G92 and R192 and [20]. its correctMoreover, orientation R192 is wouldbelieved be ensured to favor by the the adjacent sugar ring D194. rotation The conservation that is needed ofthese for C4 residues,-epimerization, and their and environment, its correct in allorientation of the UGA4Es would characterized be ensured by to the date adjacent is displayed D194. The in Figure conservation6. Crucially, of these the residues, sequence and logo their of the putativeenvironment, UGA5E in sequencesall of the UGA4Es is found characterized to be practically to date identical. is displayed In order in Figure to make 6. Crucially, a comparison the to a relatedsequence C5-epimerase, logo of the putative GM35E UGA5E from sequences the same is CEP1 found family to be practically was analyzed identical. as well. In order In addition, to make the sequencea comparison logo ofto UDP-glucosea related C5-epimerase, 4-epimerase GM35E (Gal4E), from the the most same studied CEP1 family specificity was analyzed within the as CEP1well. family,In wasaddition, also the included. sequence As logo a totallyof UDP-glucose different sequence4-epimerase logo (Gal4E), was obtainedthe most studied for the specificity latter two within the CEP1 family, was also included. As a totally different sequence logo was obtained for the epimerases, it can be concluded that the residues are not simply conserved among all CEP1 members latter two epimerases, it can be concluded that the residues are not simply conserved among all CEP1 or even among enzymes with the same epimerization site. Accordingly, G92, R192, and D194 can members or even among enzymes with the same epimerization site. Accordingly, G92, R192, and probably serve as fingerprints for the UGA4E specificity. In addition, similar differences are observed D194 can probably serve as fingerprints for the UGA4E specificity. In addition, similar differences in theare YxxxK observed catalytic in the motif YxxxK (Figure catalyti6).c Thesemotif (Figure results 6). indicate These thatresults the in putativedicate that UGA5E the putative sequences UGA5E are probablysequences just incorrectly are probably annotated just incorrectly UGA4Es. annotated UGA4Es.

Catalysts 2020, 10, 222 7 of 11 Catalysts 2020, 10, 222 7 of 11

FigureFigure 6. 6.Overview Overview of of sequence sequence logos.logos. The conservation conservation of of various various active active site site residues, residues, including including their their environment,environment, and and the the YxxxK YxxxK motif motif were were evaluated evalua forted UDP-glucuronate for UDP-glucuronate 4-epimerase 4-epimerase (UGA4E), (UGA4E), putative UDP-glucuronateputative UDP-glucuronate 5-epimerase 5-epimerase (UGA5E), (UGA5E), GDP-mannose GDP-mannose 3,5 epimerase 3,5 epimerase (GM35E), (GM35E), and UDP-glucoseand UDP- 4-epimeraseglucose 4-epimerase (Gal4E). Residues (Gal4E). areResidues numbered are numbered according according to the UGA4E to the from UGA4EStreptomyces from Streptomyces viridosporus. viridosporus. 3. Discussion 3. DiscussionCarbohydrate epimerases have recently come into focus as they are a valuable route toward rare sugar production.Carbohydrate However, epimerases more have research recently on come this enzymeinto focus class as they is required are a valuable in order route to fully toward appreciate rare theirsugar potential. production. Even However, though 39 more specificities research have on this been enzyme discovered classto is date, required in-depth in order information to fully on theseappreciate enzymes’ their physiological potential. Even roles though or catalytic 39 specificities mechanisms have is often been lacking. discovered Therefore, to date, eff in-depthorts should beinformation made to characterize on these representativesenzymes’ physiological in orderto role fills in or these catalytic knowledge mechanisms gaps. Moreover,is often lacking. prominent mechanisticTherefore, insightsefforts should might be lead made to theto characterize rational engineering representatives of these in order biocatalysts, to fill in potentially these knowledge resulting in alteredgaps. Moreover, specificities, prominent stabilities, mechanistic or activities. insights might lead to the rational engineering of these biocatalysts,On that account, potentially this resulting study focusedin altered on specificities, the UGA5E stabilities, specificity, or activities. hypothesized to catalyze the reversibleOn epimerizationthat account, this of UDP-GlcA study focused toward on UDP-IdoAthe UGA5E [ 9specificity,]. The enzyme’s hypothesized activity wasto catalyze only detected the reversible epimerization of UDP-GlcA toward UDP-IdoA [9]. The enzyme’s activity was only in rabbit tissue before, and there is no report on the recombinant expression or characterization of detected in rabbit tissue before, and there is no report on the recombinant expression or a putative UGA5E to date. This research clustered all 1154 putative UGA5E sequences from the characterization of a putative UGA5E to date. This research clustered all 1154 putative UGA5E UniProt database in a phylogenetic tree and the combination of experimental work and in silico analysis sequences from the UniProt database in a phylogenetic tree and the combination of experimental resultedwork and in the in hypothesissilico analysis that resulted these sequencesin the hypothesis actually th displayat these UGA4Esequences specificity. actually display Subsequently, UGA4E the existencespecificity. of the Subsequently, UGA5E specificity the existence in general of the was UGA5 questioned,E specificity resulting in general in a was physiological questioned, issue. resulting Indeed, it wasin a physiological previously hypothesized issue. Indeed, thatit was UGA5E previously is part hypothesized of the enzymatic that UGA5E pathway is part forof the the enzymatic production ofpathway IdoA-containing for the production DS starting of IdoA-containing from UDP-Glc DS (Figure starting7a) from [ 9]. UDP-Glc To shed (Figure more light 7a) [9]. on To this shed issue, pathwaysmore light relevant on this for issue, other pathways eukaryotic relevant and prokaryotic for other eukaryotic IdoA-containing and pr polysaccharides,okaryotic IdoA-containing e.g., heparin sulfate,polysaccharides, were examined e.g., heparin [11,14 ,sulfate,29]. These were metabolic examined [11,14,29]. pathways These start metabolic with the actionpathways of anstart UDP-Glc with dehydrogenase,the action of an converting UDP-Glc dehydrogenase, UDP-Glc into UDP-GlcA. converting However, UDP-Glc thisinto sugarUDP-GlcA. acid is However, then first this polymerized sugar beforeacid theis then resulting first polymerized polysaccharide before undergoes the result modificationsing polysaccharide such as undergoes sulfation or modifications epimerization. such In as other words,sulfation GlcA or is, epimerization. thus, epimerized In other to IdoA words, at theGlcA polysaccharide is, thus, epimerized level instead to IdoA of at at the the polysaccharide NDP-sugar level. Forlevel the bacterialinstead of GAGs at the structures NDP-sugar and level. heparin For th/heparane bacterial sulfate, GAGs this structures epimerization and heparin/heparan step is catalyzed bysulfate, an enzyme this epimerization referred to as step glucuronyl is catalyzed C5-epimerase by an enzyme (EC referred 5.1.3.17, to as CEP8) glucuronyl [30,31 ].C5-epimerase For dermatan (EC and chondroitin5.1.3.17, CEP8) sulfate [30,31]. synthesis, For dermatan it was foundand chondroitin that this reactionsulfate synthesis, is realized it was by found chondroitin-glucuronate that this reaction C5-epimeraseis realized by (EC chondroitin-glucuronate 5.1.3.19, not classified C5-epimerase in a CEP family) (EC [ 325.1.3.19,,33]. not classified in a CEP family) [32,33]. These results thus contradict the hypothesis of Jacobson et al., although the outcomes can be These results thus contradict the hypothesis of Jacobson et al., although the outcomes can be unified [9]. Indeed, the detected UDP-glucose dehydrogenase activity is anyway present as the unified [9]. Indeed, the detected UDP-glucose dehydrogenase activity is anyway present as the first first step in the pathway in order to obtain UDP-GlcA. Subsequently, the assumed UGA5E activity step in the pathway in order to obtain UDP-GlcA. Subsequently, the assumed UGA5E activity can be canexplained be explained by the by fact the that fact they that analyzed they analyzed protein fractions protein fractionsand not purified and not proteins. purified The proteins. detected The detectedUGA5E UGA5E activity activityin such in a such fraction a fraction was, was,thus, thus,most mostprobably probably the thecombined combined action action of a of a glycosyltransferaseglycosyltransferase that that catalyzescatalyzes thethe polymerizationpolymerization and and chondroitin-glucuronate chondroitin-glucuronate C5-epimerase, C5-epimerase, resulting in IdoA subunits (Figure7b). Subsequently, the polysaccharide can be further sulfated, Catalysts 2020, 10, 222 8 of 11 Catalysts 2020, 10, 222 8 of 11 eventuallyresulting in yielding IdoA subunits DS. In general, (Figure the7b). probabilitySubsequently, of thethe presencepolysaccharide of the UGA5Ecan be further specificity sulfated, in nature iseventually further decreased yielding DS. by In the general, observation the probability that no additionalof the presence functions of the UGA5E for free specificity (UDP-)l-iduronic in nature acid haveis further been decreased detected toby date the observation and no extra that evidence no additional on this functions epimerase for specificityfree (UDP-) hasL-iduronic been published acid sincehave 1962.been detected to date and no extra evidence on this epimerase specificity has been published sinceIn 1962. conclusion, this research states that the assumed UGA5E specificity present in sequence databasesIn conclusion, is not justified, this research and that states the correspondingthat the assumed EC UGA5E entry should specificity be retracted. present in The sequence epimerase databases is not justified, and that the corresponding EC entry should be retracted. The epimerase specificity was initially studied as a potential route toward IdoA production, which can then be used specificity was initially studied as a potential route toward IdoA production, which can then be used for the synthesis of heparin. On that account, an enzymatic route comprising chondroitin-glucuronate for the synthesis of heparin. On that account, an enzymatic route comprising chondroitin-glucuronate C5-epimerase or glucuronyl C5-epimerase can still be exploited. In addition, this study unlocks a C5-epimerase or glucuronyl C5-epimerase can still be exploited. In addition, this study unlocks a new newpool pool of UGA4E of UGA4E sequences, sequences, which which can canbe deployed be deployed for UDP-GalA for UDP-GalA synthesis, synthesis, and andhas highlighted has highlighted fingerprintsfingerprints thatthat could be used used for for the the correct correct annotation annotation of ofsequences sequences harboring harboring UGA4E UGA4E activity. activity.

Figure 7. Production of dermatan sulfate. (a) Hypothesized pathway containing the putative UGA5E speciﬁcityFigure 7. Production versus (b) conﬁrmedof dermatan pathway sulfate. ( ona) Hypothesized a polysaccharide pathway level. cont Glc:aining glucose; the GlcA:putative glucuronic UGA5E acid; GalNAc:specificityN versus-acetyl- (db-galactosamine;) confirmed pathway IdoA: onl-iduronic a polysaccharide acid; DH: level. UDP-glucose Glc: glucose; dehydrogenase; GlcA: glucuronic UGA5E: UDP-glucuronateacid; GalNAc: N-acetyl- 5-epimerase;D-galactosamine; GT: glycosyltransferase; IdoA: L-iduronic C5-epi: acid; chondroitin-glucuronateDH: UDP-glucose dehydrogenase; C5-epimerase. UGA5E: UDP-glucuronate 5-epimerase; GT: glycosyltransferase; C5-epi: chondroitin-glucuronate C5- 4. Materialsepimerase. and Methods

4.1.4. Materials Materials and Methods All chemicals were obtained from Sigma-Aldrich (Saint Lois, MO, USA) or Carbosynth (Compton, 4.1. Materials UK) and were of the highest purity, unless stated otherwise. All chemicals were obtained from Sigma-Aldrich (Saint Lois, MO, USA) or Carbosynth 4.2.(Compton, Gene Cloning UK) and and were Transformation of the highest purity, unless stated otherwise. The codon-optimized putative UGA5E genes from Thermodesulfobacterium geofontis (UniProt: 4.2. Gene Cloning and Transformation F8C4X8) and Agrobacterium radiobacter strain K84 (UniProt: B9J8R3) were synthesized and subcloned The codon-optimized putative UGA5E genes from Thermodesulfobacterium geofontis (UniProt: into the pET21 vector at NdeI and XhoI restriction sites, providing a C-terminal His6-tag, by GeneArt GeneF8C4X8) Synthesis and Agrobacterium (Thermo Fisher radiobacter Scientiﬁc, strain Waltham, K84 (UniProt: MA, B9J8R3) USA). Thewere constructs synthesized were and transformedsubcloned in into the pET21 vector at NdeI and XhoI restriction sites, providing a C-terminal His6-tag, by GeneArt E. coli BL21(DE3) electrocompetent cells for protein expression. Gene Synthesis (Thermo Fisher Scientific, Waltham, MA, USA). The constructs were transformed in 4.3.E. coli Enzyme BL21(DE3) Production electrocompetent cells for protein expression.

4.3. First,Enzyme 250 Production mL lysogeny broth (LB) medium (10 g/L trypton, 5 g/L yeast extract and 5 g/L NaCl) supplemented with 100 µg/mL ampicillin was inoculated with 2% (v/v) stationary culture and incubated First, 250 mL lysogeny broth (LB) medium (10 g/L trypton, 5 g/L yeast extract and 5 g/L NaCl) at 37 C and 200 rpm until the OD equaled 0.6. Subsequently, enzyme production was induced supplemented◦ with 100 µg/mL ampicillin600 was inoculated with 2% (v/v) stationary culture and by the addition of isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 1 mM and incubated at 37 °C and 200 rpm until the OD600 equaled 0.6. Subsequently, enzyme production was theinduced culture by was the addition incubated of overnightisopropyl β at-D-thiogalactopyranoside 20 ◦C and 200 rpm. Cells (IPTG) were to harvested a final concentration by centrifuging of 1 for 30 min at 9000 rpm and 4 C. The obtained pellet was frozen and stored at 20 C for at least 1 h. mM and the culture was◦ incubated overnight at 20 °C and 200 rpm. Cells− were◦ harvested by centrifugingIn order for to 30 obtain min at crude 9000 cellrpm lysateand 4 °C. for The the obtained initial specificity pellet was tests,frozen the and pellet stored was at –20 resuspended °C for inat 8 least mL 1 of h. lysis buffer (10 mM imidazole and 100 µM phenylmethane sulfonyl fluoride (PMSF) in 100 mMIn order MOPS to pH obtain 7.5) crude and cooled cell lysa onte ice for for the 30 initial min. specificity Subsequently, tests,the the cellspellet were was furtherresuspended disrupted in by sonication8 mL of lysis (3 timesbuffer 2.5 (10 min, mM Bransonimidazole sonifier and 100 250, µM level phenylmethane 3, 50% duty sulfonyl cycle). fluoride (PMSF) in 100 On the other hand, purified enzyme was obtained by first lysing the cells by resuspending the pellet in 8 mL of lysis buffer (500 mM NaCl, 10 mM imidazole, 100 µM PMSF, and 1 mg/mL lysozyme Catalysts 2020, 10, 222 9 of 11 in 50 mM sodium phosphate buffer pH 7.4) and cooling them on ice for 30 min. Next, the cells were subjected to 3 times 2.5 min of sonication (Branson sonifier 250, level 3, 50% duty cycle). Cell debris was collected by 15 min of centrifugation at 12000 rpm and 4 ◦C. Next, the His6-tagged proteins present in the supernatant were purified to apparent homogeneity by nickel-nitrilotriacetic acid chromatography, with small variations to the supplier’s description (Thermo Scientific, Waltham, MA, USA). The resin was washed twice with 8 mL wash buffer (500 mM NaCl, 20 mM imidazole in 50 mM sodium phosphate buffer pH 7.4). Protein was eluted with 3 times 4 mL elution buffer (500 mM NaCl, 250 mM imidazole in 50 mM sodium phosphate buffer pH 7.4). As a final step, buffer was exchanged to 100 mM MOPS pH 7.5 by using Amicon Ultra-15 centrifugal filter units with 30 kDa cut-off (Merck Millipore, Darmstadt, Germany). For TgUGAE, the purification yield was only 0.2 mg/L culture, whereas for ArUGAE, this was about 45 mg/L (Figures S7 and S8). The protein content was determined by measuring the absorbance at 280 nm with the NanoDrop2000 Spectrophotometer (Thermo Scientific, Waltham, MA, USA). The extinction coefficient and molecular weight of His6-tagged enzymes were calculated using the ProtParam tool on the ExPASy server (https://web.expasy.org/protparam/). Molecular weight and purity of the protein were verified by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE; 12% gel). As the expression of TgUGAE was low, the measured protein concentration was adjusted using the ImageJ software [34].

4.4. Activity Testing For the examination of the putative UGA5Es, 10 mM UDP-GlcA was incubated together with 100 µL crude cell lysate in 100 mM MOPS pH 7.5 at 30 ◦C (500 µL total reaction volume). A substrate control (10 mM UDP-GlcA in 100 mM MOPS pH 7.5), as well as an enzyme control (100 µL crude cell lysate in 100 mM MOPS pH 7.5) were included and treated in the exact same way as the reaction mixture. Tests were performed in triplicate. In addition, the potential activity of the background proteins was tested by setting up a reaction with 10 mM UDP-GlcA and 100 µL crude cell lysate obtained from induced E. coli BL21(DE3) cells containing an empty pET21 vector in 100 mM MOPS pH 7.5 at 30 ◦C. The influence of NAD+ on the enzyme’s specificity was tested by incubating 0.1 mg/mL enzyme with + 10 mM UDP-GlcA and 5 mM NAD in 100 mM MOPS pH 7.5 at 30 ◦C (400 µL total reaction volume). The enzymes’ specific activities were determined with 10 mM UDP-GlcA and 0.05 mg/mL purified enzyme in a total reaction volume of 300 µL 100 mM MOPS pH 7.5 at 30 ◦C. For all activity tests, samples were taken at defined time points and inactivated in 90 mM acetic acid. The samples were then hydrolyzed for 1 h at 95 ◦C in order to obtain the respective monosaccharides. The hydrolyzed reaction mixtures were analyzed using high-performance anion exchange chromatography and pulsed amperometric detection (HPAEC-PAD; Dionex ICS-3000 system, Thermo Fisher Scientific, Waltham, MA, USA). The separation of the various uronic acids was realized by an isocratic method using a mixture of 150 mM NaOAch and 100 mM NaOH for 10 min.

4.5. Sequence Analysis All amino acid sequences annotated as ‘UDP-glucuronate 5-epimerase’ were retrieved from the UniProt database (https://www.uniprot.org) and subsequently aligned with Clustal Omega using default parameters (https://www.ebi.ac.uk/Tools/msa/clustalo/)[35]. The concomitant phylogenetic tree was visualized with iTOL (https://itol.embl.de). BLAST analyses were performed at the UniProt server using default parameters. For the sequence logos, the sequences of the characterized UGA4Es, GM35Es, and Gal4Es were extracted from the UniProt database and aligned with Clustal Omega using default parameters. Subsequently, the sequence logos were generated by WebLogo [36].

Supplementary Materials: The following are available online at http://www.mdpi.com/2073-4344/10/2/222/s1, Figure S1. Optimization of the recombinant expression of TgUGAE; Figure S2. Analysis of potential UGA4E activity in the E. coli background; Figure S3. Chromatographic analysis of the reaction controls for TgUGAE; Figure S4. Optimization of ArUGAE overexpression in E. coli; Figure S5. Exclusion of potential UGA4E activity in the E. coli background; Figure S6. Chromatographic analysis of the reaction controls for ArUGAE; Figure S7. SDS-PAGE analysis of TgUGAE puriﬁcation; Figure S8. SDS-PAGE analysis of ArUGAE puriﬁcation. Catalysts 2020, 10, 222 10 of 11

Author Contributions: Conceptualization, O.G., T.D., S.V.O., M.D.C. and K.B.; methodology, O.G.; validation, S.V.O., M.D.C., T.D. and K.B.; formal analysis, O.G.; investigation, O.G.; data curation, O.G.; writing—original draft preparation, O.G.; writing—review and editing, T.D., S.V.O., M.D.C. and K.B.; visualization, O.G.; supervision, T.D. and K.B.; project administration, O.G. All authors have read and agree to the published version of the manuscript. Funding: This research was funded by the Fund for Scientific Research Flanders (FWO-Vlaanderen) via a doctoral scholarship for O.G. (grant n◦ 1S16817N), S.V.O. (grant n◦ FWO.3F0.2016.0056.01) and M.D.C. (grant n◦ 1S03020N), as well as the EpiSwitch project (grant n◦ G0F3417N). Conflicts of Interest: The authors declare no conflict of interest.

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