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Probing and Engineering Key Residues for Bis-Cglycosylation and Promiscuity of a C-glycosyltransferase Dawei Chen, Shuai Fan, Ridao Chen, Kebo Xie, Sen Yin, Lili Sun, Jimei Liu, Lin Yang, Jianqiang Kong, Zhaoyong Yang, and Jungui Dai ACS Catal., Just Accepted Manuscript • DOI: 10.1021/acscatal.8b00376 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 2018

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ACS Catalysis

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Probing and Engineering Key Residues for Bis-C-

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glycosylation and Promiscuity of a C-

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glycosyltransferase

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Dawei Chen,† Shuai Fan,‡ Ridao Chen,† Kebo Xie,† Sen Yin,† Lili Sun,§ Jimei Liu,† Lin Yang,§

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Jianqiang Kong,† Zhaoyong Yang,‡ and Jungui Dai†,*

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†

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Materia Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, 1

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Xian Nong Tan Street, Beijing 100050, China.

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‡

Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union

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State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of

Medical College, 1 Tian Tan Xi Li, Beijing 100050, China. §

College of Life and Environmental Sciences, Minzu University of China, 27 Zhong Guan Cun Southern Street, Beijing 100081, China

ACS Paragon Plus Environment

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ABSTRACT

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C-glycosyltransferases (CGTs) are powerful tools for the C-glycosylation of natural and

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unnatural products. However, CGTs able to catalyze bis-C-glycosylation are very rare and the

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key amino acids of which are not uncovered. Here, we discovered a C-glycosyltransferase

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MiCGTb from Mangifera indica, which has the capacity for bis-C-glycosylation. Further studies

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on active-site motifs revealed that I152 of MiCGTb was the critical amino acid residue for the

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second C-glycosylation and its S60/V100/T104 were the pivotal residues for bis-C-glycosylation

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activity. Moreover, we developed a panel of variants with acceptor and donor promiscuity by

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site-directed mutagenesis. Among these variants, a mutant MiCGT-E152L displayed a broader

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acceptor scope for bis-C-glycosylation, and three mutants of MiCGTb exhibited sugar donor

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promiscuity towards structurally varied α-D- and β-L-glycosyl donors. Our work provides

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insights into the pivotal amino acid residues of CGTs for bis-C-glycosylation and biocatalytic

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tools to efficiently produce structurally diverse bis-C-glycosides with two identical or different

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sugar moieties in drug discovery.

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KEYWORDS

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Bis-C-glycosylation; C-glycosides; enzyme catalysis; site-directed mutagenesis; transferases

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INTRODUCTION

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Bis-C-glycosyl compounds have been isolated from both plants and microbes and have

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attracted strong interest due to their potential pharmaceutical activity and pronounced stability in

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drug development.1–3 The most notable structural feature is the two identical or different C-


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ACS Catalysis

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glycosyl moieties at different positions of “aglycone” molecules. Nevertheless, naturally

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occurring bis-C-glycosides and their structural diversity appear to be comparatively sparse,

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making it necessary to further explore their synthesis and pharmaceutical applications. However,

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the chemical synthesis of bis-C-glycosides bearing two identical or different sugar moieties in

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high yields still encounters challenges.3–8 The C-glycosyltransferases (CGTs), as promising

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alternatives to catalyze the formation of bis-C-glycosides, have attracted increasing interest and

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achieved progress in the bis-C-glycosylation of both natural and unnatural flavones by different

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CGTs (OsCGT from Oryza sativa catalyzed the first C-glycosylation with UDP-α-D-glucose,

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UDP-Glc; Desmodium incanum root proteins catalyzed the second C-glycosylation with UDP-

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Glc, UDP-α-D-galactose or UDP-β-L-arabinose).9–11 However, the CGTs naturally catalyzing

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both the first and second C-glycosylations by one CGT have only been identified from citrus

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plants,12 which displayed relative substrate specificity for bis-C-glycosylation (such as 2-

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hydroxynaringenin, phloretin and 2-phenyl-2ʹ,4ʹ,6ʹ-trihydroxyacetophenone), thus limiting their

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availability and scope to produce structurally diverse C-glycosides. To our knowledge, these are

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also the only known example of CGTs that were able to catalyze the second C-glycosylation of

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mono-C-glycosides.12 Bechthold and colleagues first revealed the primary region of a bacterial

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CGT that determines the mono-C-glycosylation by targeted-amino acid swapping studies,13 and

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the structural features critical for differentiating between C- and O-glycosyl transfer were

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demonstrated by Einsle.14 Nidetzky and co-workers also switched the glycosidic bond-type

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specificity within a homologous pair of plant mono-CGT and OGT through exchange of active-

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site motifs.15 Moreover, the key residues of CGTs for mono-C-glycosylation have been explored

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by protein modeling and site-directed mutagenesis studies.16,17 However, little is known about

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the determinant residues of CGTs for bis-C-glycosylation. Therefore, it is greatly desired to


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probe key amino acids of CGTs for the second C-glycosylation and expand their substrate scope

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to generate more novel structurally diverse bis-C-glycosides.

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Scheme 1. C-glycosylation of 1 by MiCGTb (a) and MiCGT (b)

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We recently identified two novel benzophenone CGTs, MiCGT and MiCGTb, from Mangifera

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indica, sharing 90% sequence identity.18,19 They are suggested to be involved in the biosynthesis

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of mangiferin, a xanthonoid C-glucoside of various pharmacological potential, including

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antioxidant and antidiabetic activity.20–22 Thus, further investigation of the catalytic potential of

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MiCGT and MiCGTb could be useful in the pharmacological applications. Interestingly, we

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found that MiCGTb can catalyze the first C-glycosylation and successive second C-glycosylation

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of 2-phenyl-2',4',6'-trihydroxyacetophenone (1) with UDP-Glc to yield bis-C-glucosyl compound

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(1a), while MiCGT only produces the mono-C-glucosyl derivative (1b) (Scheme 1). This finding

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inspired us to examine the functional divergence of MiCGT and MiCGTb with such high

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sequence identity. Furthermore, understanding the factors governing this unique second C-

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glycosylation would facilitate further rational design for more novel promiscuous CGT variants.

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Herein, we report the exploration of determinant residues that control the second C-glycosylation

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of MiCGTb and the derivation of a panel of MiCGTb and MiCGT variants with robust acceptor

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and donor promiscuity by protein engineering.

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RESULTS

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I152 is the key residue of MiCGTb for the second C-glycosylation. Owing to the limited

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structural information available on plant CGTs, our initial studies focused on the construction of


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ACS Catalysis

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MiCGT-MiCGTb chimeras through exchange of active-site motifs. By analyzing the sequence

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alignment of MiCGT and MiCGTb, we found that their differential amino acids were distributed

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in the N-terminal domains, which were divided into five regions, A–E, based on their predicted

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secondary structure differences (Figure 1a). To identify key sequences in MiCGTb for the

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second C-glycosylating function, we designed and generated ten chimeras (MiCGTb-A/B/C/D/E,

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MiCGT-A/B/C/D/E) by reciprocally exchanging the targeted residues for mutation in the protein

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regions A–E (Table S1). Of the five chimeras of MiCGTb, only MiCGTb-D was dramatically

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impaired in its ability for the second C-glycosylation of 1 (Figure S1a). However, the reverse

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engineering of MiCGT revealed that only the corresponding chimera MiCGT-D acquired the

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capability for the second C-glycosylation (Figure S1b). These results unequivocally suggested

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that the D region of MiCGTb was crucial to the second C-glycosylation.

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To assign the key residues in the D region, we swapped the single amino acid between the two

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enzymes (Table S1). The enzyme assay by HPLC-MS2 revealed that the mutant MiCGT-E152I

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gained the ability to catalyze the second C-glycosylation of 1, whereas the mutant MiCGTb-

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I152E almost lost this function because of a single point mutation (Figure 1b). Moreover, the

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single point mutation in 152 had little effect on the mono-C-glycosylation of MiCGT and

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MiCGTb. Altogether, these data suggest that position 152 is the critical residue for the second C-

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glycosylation. Subsequently, position 152 was targeted for site-directed mutagenesis to rationally

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design more promiscuous CGTs. We obtained nineteen MiCGT mutants by site-saturation

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mutagenesis of position 152, fourteen of which were endowed with the capacity for bis-C-

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glycosylation (Figure S2). Notably, seven MiCGT mutants with isoleucine, methionine, valine,

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asparagine, threonine, alanine or leucine led to high yields of 1a with respect to MiCGTb (Table

23

S2), whereas no bis-C-glucoside formation was observed when residue 152 was mutated to


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proline, arginine, aspartate, tryptophan or tyrosine. Similarly, MiCGTb mutants with methionine,

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valine or threonine were detected with enhanced activity for the second C-glycosylation of 1, and

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the I152M mutant showed the highest activity (Figure S3).

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Figure 1. Secondary structure-based sequence alignment and percent conversion of 1a by

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MiCGTb and MiCGT mutants. (a) The secondary structure elements of MiCGT and MiCGTb

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(N-domain region, residues 50–230) were predicted by the JPred4 program. The red rectangles

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indicate α-helix, the thick black arrows indicate β-sheet. The asterisks indicate targeted positions

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for mutagenesis; (b) Percent conversion of 1a by HPLC of MiCGT and MiCGTb mutants with

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single amino acid exchanges in the D region. N.D.: not detected. WT: wild type.

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Improving bis-C-glycosylation activity of MiCGTb by site-directed mutagenesis. During

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exploration of the determinant region for the second C-glycosylation, we also observed that the

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conversion rates of 1a of chimera MiCGTb-B was increased by approximately 2 times compared

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with the wild-type MiCGTb, whereas that of chimera MiCGTb-A was reduced by 30% (Figure

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S1a). Thus, the regions A and B in MiCGTb were crucial to the bis-C-glycosylation activity.


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ACS Catalysis

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Moreover, the impact of B region on the bis-C-glycosylation activity was greater than that of A

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region. To locate the key residues within these two regions in MiCGTb, the targeted single

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amino acid in each region of MiCGTb was replaced by the corresponding residue in MiCGT,

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leading to five mutants (Table S1). The subsequent enzyme assay by HPLC-MS2 revealed that

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the MiCGTb-S60L mutant reduced the bis-C-glycosylation activity, and both MiCGTb-V100A

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and T104N mutants resulted in increased activity (Figure S4). Similarly, these above mutants of

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MiCGTb displayed >99% conversion rates for mono-C-glycosylation. Thus, these data

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suggested the pivotal contribution of these three positions to the catalytic activity for the second

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C-glycosylation of MiCGTb. To rationally produce more promiscuous and catalytically efficient

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CGTs, their site-saturation mutagenesis were further performed individually. The conversion

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rates of the mutants with glycine/lysine (site 60) were 71% and 81%, respectively (Figure S5);

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the conversion rates of the mutants with alanine/serine (site 100) were 87% and 67%,

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respectively (Figure S6); the conversion rates of the mutants with asparagine/glycine (site 104)

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were 51% and 68%, respectively (Figure S7). Therefore, these residues were the optimal amino

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acids for each position with higher activity. Combined with site 152, we generated eight

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quadruple mutants of MiCGTb based on the templates of the above mutations at position 60, 100

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and 104 (Table S3). As expected, the bis-C-glycosylation activities of these eight mutants were

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all enhanced, especially the mutants MiCGTb-GAGM and MiCGTb-KAGM with approximately

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4.2-fold for 1 with respect to the wild-type enzyme (Figure S8).


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Figure 2. Sugar acceptor promiscuity of MiCGT and MiCGTb mutants for bis-C-glycosylation.

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(a) Structures of sugar acceptors (1–14); (b) Percent conversion of bis-C-glycosides by enzyme

4

mutants with UDP-Glc (16). The members are listed based on the structural scaffolds shown in

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part a. Bis-C-glucosylated products (1a–3a) were prepared as described in Experimental Section

6

and structurally confirmed by MS, and 1H and 13C NMR spectroscopy.

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Biochemical characteristics of CGT variants. As described above, fifteen CGT variants with

8

increased activity, including seven MiCGT-E152 mutants (Table S2) and eight quadruple

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mutants of MiCGTb (Table S3) were accordingly selected to further investigate their

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biochemical characteristics. Their pH preference was determined using 1 and UDP-Glc (16) as

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acceptor and donor substrates, with all enzymes exhibiting higher conversion rates of 1a at

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pH7.0–10.0 (Figure S9). The kinetic parameters of these enzymes were calculated using 1 and its


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ACS Catalysis

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mono-C-glucoside 1b as substrates, respectively. The kcat/Km values for the first C-glycosylation

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of 1 were 105~106 M-1min-1, whereas those corresponding values for the second C-glycosylation

3

were only 102~103 M-1min-1 (Table S4). These results clearly demonstrate that the catalytic

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efficiency of the first C-glycosylation of 1 is two or more orders of magnitude faster than that of

5

the second C-glycosylation, which might be due to the hindrance of attached sugar groups.23

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Thus the latter C-glycosylation was the rate-limiting step in the sequential bis-C-glycosylation.

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Notably, the catalytic efficiency of MiCGT-E152M and MiCGTb-KSGM mutants for the second

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C-glycosylation were each enhanced approximately 5.6- and 3.2-fold higher than that of

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MiCGTb.

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Acceptor promiscuity of CGT variants for bis-C-glycosylation. To explore the synthetic

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usefulness of these fifteen mutants above in vitro, an acceptor library of 14 representative natural

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and unnatural compounds that could be subjected to the wild-type MiCGT or MiCGTb catalysis

13

for mono-C-glycosylation (Figure 2a), was assessed with UDP-Glc. An initial hint concerning

14

the enzymes’ unusual broad capability for bis-C-glycosylation was manifested by HPLC-

15

UV/MS2 analysis (Figure 2b and Figures S10–S20), which revealed that these mutants were

16

sufficiently flexible to bis-C-glycosylate in at least six library members. Remarkably, the

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MiCGT-E152L mutant displayed bis-C-glycosylation activity towards thirteen substrates with

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one exception of 12, whereas the wild-type MiCGT had no ability for the second C-glycosylation

19

towards all acceptors. Moreover, high bis-C-glycosylation conversion rates (>60%) were

20

observed with seven substrates (1–5, 10 and 13). Thus, the acceptor promiscuity and catalytic

21

efficiency of enzymes for the second C-glycosylation were significantly enhanced through

22

engineering. Notably, we found that the seven MiCGT-derived mutants, with just one amino acid

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difference at position 152, exhibited significant differences in the substrate scope and catalytic


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activity (Figure 2b), which could be exploited for the directed synthesis of bis-C-glycosides. In

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addition, the eight MiCGTb mutants not only recognized 3'-glucosyl phloretin (2b) but also

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catalyzed 3'-dimethylallyl phloretin (15) to form C-glucoside (15a). Interestingly, these two bis-

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C-alkylation products can undergo spontaneous oxidation to afford quinol C-glucosides (2c and

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15b) at pH 8.0 (Figure S21), respectively, thus expanding the structural diversity.24–26

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Figure 3. Sugar donor promiscuity of three MiCGTb mutants for mono-C-glycosylation. (a)

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Structures of sugar donors (16–27); (b) Percent conversion of MiCGTb, MiCGTb-GANM,

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MiCGTb-GAGM and MiCGTb-GSGM towards sugar donors in part a with 1. N.D.: not

10

detected. The mono-C-glycosides (1b–1h) were prepared as described in Experimental Section

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and structurally confirmed by MS, and 1H and 13C NMR spectroscopy. The structures of mono-

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C-glycosides are shown in Table 1.

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Sugar donor promiscuity of CGT variants. Subsequently, the sugar donor promiscuity of

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the fifteen CGT mutants was probed using a dozen NDP-sugars (16–27; Figure 3a) in the

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presence of 1 as the acceptor. This panel consisted of UDP-α-D-glucose (16), TDP-α-D-glucose


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ACS Catalysis

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(17), UDP-α-D-galactose (18), UDP-β-L-rhamnose (19), UDP-β-L-arabinose (20), UDP-α-D-N-

2

acetyl-glucosamine (21), UDP-α-D-xylose (22), UDP-α-D-glucuronic acid (23), UDP-β-L-

3

fucose (24), UDP-α-D-N-acetyl-galactosamine (25), GDP-α-D-mannose (26) and UDP-α-D-N-

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acetyl-gulosamine (27), cumulatively representing the hexose and pentose with α-D- or β-L-

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configuration, and various functional moieties at C-1, C-2, C-3, C-4 or/and C-6 with different

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substituents. To our delight, the eight MiCGTb mutants exhibited sugar donor promiscuity and

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high conversion rates (>70%) for the first C-glycosylation towards eight of twelve sugar

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nucleotide donors, including six D-sugars and two L-sugars (Figures S22–S25). Notably, three

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MiCGTb mutants (GANM/GAGM/GSGM) displayed mono-C-glycosylation activity on eleven

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sugar donors (16–26) with one exception of 27 (Figure 3b), reasserting their broad substrate

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scope, while the wild-type MiCGTb could recognize six sugar donors (16–20 and 22). Moreover,

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MiCGTb mutants were able to catalyze α- and β-glycosyl donors to form β- and α-glycosides in

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the inverting mechanism, respectively. Interestingly, the resulting respective mono-C-glycosides

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(1b–1h) with various D- or L-glycosyl moieties have also functioned as glycoside acceptors for

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the second C-glycosylation with 16 to generate bis-C-glycosides with two different sugar

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moieties (Table 1; Figures S26–S31). In contrast, the mono-C-glucoside (1b) could not be

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catalyzed by these three MiCGTb mutants with other NDP-sugar donors (18–26). The sequential

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C-glycosylation with NDP-sugars (16–26) and C-glycosylation with UDP-Glc (16) might be due

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to their preference for UDP-Glc further supported by the higher Km but lower kcat/Km values of

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MiCGTb-GAGM towards different UDP-sugars (18, 19, 21 and 23) compared with UDP-Glc

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(Table S5). Furthermore, with respect to the wild-type enzyme, the three MiCGTb mutants not

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only have a 3-fold higher bis-C-glycosylation activity for the mono-C-glycosides (1b–1e and 1g)


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but could also recognize mono-C-N-acetylglucoside (1f) and mono-C-glucuronide (1h) as

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acceptors (Table 1; Figures S30 and S31).

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Table 1. Percent conversion of bis-C-glycosides (1a and 1ca–1ha) by MiCGTb, MiCGTb-

4

GANM, MiCGTb-GAGM and MiCGTb-GSGM with mono-C-glycosides (1b–1h) and UDP-Glc

5

(16)a

6 Conversion / % Sub.

7 8 9

MiCGTb

MiCGTbGANM

MiCGTb -GAGM

MiCGTb -GSGM

1b

34

86

88

80

1c

23

94

95

100

1d

43

100

100

100

1e

40

100

100

100

1f

0

27

24

23

1g

45

100

100

100

1h

0

32

40

39

a

The bis-C-glycosides (1ca, 1da and 1ga) were prepared as described in Experimental Section and structurally confirmed by MS, and 1H and 13C NMR spectroscopy. The structures of bis-Cglycosides are shown in Figure S28.

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DISCUSSION

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Studies on microbial and plant CGTs have attracted considerable interest and achieved great

13

progress in the enzymatic mono-C-glycosylation of natural and unnatural products such as


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ACS Catalysis

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flavone,9–11,27–31 benzophenone and xanthone,18,19 and anthraquinone.32–36 However, only a report

2

from Taguchi and colleagues demonstrated that a single enzyme can catalyze bis-C-

3

glycosylation reactions with UDP-Glc.12 Structure–function relationships of CGTs catalyzing

4

mono- and bis-C-glycosylation, in comparison to that of CGTs catalyzing mono-C-glycosylation

5

alone, are not known. In the present study, we identified that I152 was the critical residue for the

6

second C-glycosylation of a C-glycosyltransferase, MiCGTb. Specifically, the acceptor scope of

7

MiCGT for bis-C-glycosylation was dramatically expanded resulting from a single point

8

mutation at position 152. It has been reported that OGTs with higher efficiency showed the more

9

relaxed substrate specificity compared to that of the lower ones.37,38 Thorson and colleagues used

10

directed evolution to improve the substrate promiscuity of an OGT through screening efficiency

11

toward an acceptor substrate.39 Interestingly, in our work, the acceptor and donor promiscuity

12

and catalytic efficiency of MiCGTb were also significantly enhanced only by screening for

13

efficiency toward 1 and UDP-Glc. These results lend strong support to the observation that an

14

increase in enzyme proficiency leads to an increase in promiscuity,40 and provided the potential

15

to create promiscuous CGT variants simply by screening for efficiency toward a single acceptor-

16

donor pair.

17

The reaction mechanism of bacterial CGT has been intensely studied, especially that of

18

UrdGT2 from Streptomyces fradiae, in which D137 served as a catalytic base to accept an

19

aromatic proton, thereby increasing the reactivity of the phenolic acceptor in a Friedel-Crafts-

20

like reaction with the sugar donor.14,41 However, the enzymatic mechanism of plant CGTs for C-

21

glycosylation remains to be elucidated. Hirade et al. concluded from analysis of homology

22

modeling and mutagenesis that the two amino acid residues of UGT708D1 located in the active

23

site, Asp85 and Arg292, are important for C-glucosylation activity.17 Moreover, the highly


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conserved N-terminal histidine residue has been suggested as a catalytic base to deprotonate the

2

aromatic carbon of the acceptor, which undergoes a nucleophilic attack on the anomeric carbon

3

of the sugar donor.15,17 However, our homology modeling and mutagenesis studies demonstrated

4

that H23 in MiCGTb and MiCGT-E152M serves as an active-site residue for the second C-

5

glycosylation (Figure S32).41,42 Molecular dynamics (MD) simulations (Figure S33) and

6

substrate docking of MiCGT, MiCGTb and their mutants MiCGT-E152I and MiCGTb-I152E

7

were performed with ligand 1b and UDP-Glc (16). In the homology modeling, residue 152 is

8

situated at the bottom of the acceptor binding pocket. Replacement of E152 with isoleucine in

9

MiCGT extended the width of the binding pocket from 6.8 Å to 13.8 Å and created sufficient

10

space for the glycoside acceptor to interact with the sugar donor (Figures 4a and 4b), thus

11

facilitating the nucleophilic attack of C–5' of the acceptor on the anomeric carbon of the sugar

12

donor to form the bis-C-glycoside.15,17 Thus, the acceptor promiscuity of MiCGT variants might

13

be due to the increasing space of active site pocket for larger mono-C-glycosides. However, the

14

width of the binding pocket of the I152E mutant was slightly changed relative to that of

15

MiCGTb. The most striking difference between the wild-type MiCGTb and its I152E mutant is

16

the rotated orientation of ligand 1b, increasing the distance between the C–5' of the acceptor and

17

catalytic base H23 (Figures 4c and 4d), thereby disfavoring the activation of this attacking

18

carbon.15–17,43–45 Thus, it is reasonable to hypothesize that the bis-C-glycosylation reactions are a

19

consequence of creating more space for large mono-C-glycosides. However, molecular insight

20

into the precise mechanism of the bis-C-glycosylation will require further structural biology

21

investigation in the future.


14

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ACS Catalysis

1 2

Figure 4. Structure homology modeling of MiCGT and MiCGTb mutants. The structure

3

homology modeling of MiCGT (a) and E152I mutant (b), and MiCGTb (c) and I152E mutant (d)

4

after equilibrated structure docking with acceptor 1b and donor UDP-Glc (16). Residue 152 is

5

shown in red, H23 is shown in cyan. The above structures were all solvated using TIP3P water

6

molecules followed by energy minimization and 20 ns molecular dynamics simulations to

7

remove bad contacts.

8

The sugar donor promiscuity of microbial OGTs46–52 and plant OGTs53–55 has been discussed

9

within structurally varied D- and L-sugars. However, only two bacterial CGTs have been

10

reported to be able to accept both D- and L-glycosyl donors.56,57 Moreover, the known plant

11

CGTs were able to catalyze the C-glycosylation with D-sugars alone, such as D-glucose, D-

12

xylose as well as D-galactose.12,18,19,27–31 It is noteworthy that the MiCGTb mutants reported in

13

this study can tolerate the epimerized or deoxygenated sugar at C-2, C-4 or C-6, serving as the

14

potential biocatalysts for further C-glycodiversification with more natural and unnatural NDP-

15

sugars.58–61 Moreover, the MiCGTb mutants are not only able to transfer D-sugars, such as D-

16

glucose, D-galactose, D-xylose etc. to acceptor 1, but also able to accept L-sugars, such as L-


15

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Page 16 of 34

1

rhamnose, L-arabinose and L-fucose. To the best of our knowledge, such ability on plant CGTs

2

for C-glycosylation with both D- and L-glycosyl donors is reported for the first time. In addition,

3

β- and α-glycosides were produced by MiCGTb mutants with α- and β-glycosyl donors in the

4

inverting mechanism, respectively. Most importantly, our work provides a panel of CGTs that

5

were able to efficiently produce structurally diverse bis-C-glycosides with two identical or

6

different sugar moieties. These results clearly showed that the CGT variants exhibited broader

7

substrate (acceptor and donor) promiscuity and higher catalytic efficiency, especially towards D-

8

and L-sugar donors, rendering them promising catalysts for the construction of C-glycoside

9

libraries that exhibit structural and bioactive diversities. Circular dichroism spectra analyses

10

indicate that MiCGTb and the mutant MiCGTb-GAGM were folded with similar secondary

11

structure contents (Figure S34a). The fluorescence intensity of MiCGTb-GAGM exhibited 2-fold

12

enhancement relative to that of the wild-type MiCGTb, suggesting the conformational alteration

13

of protein (Figure S34b), which might be related to the increased efficiency and substrate

14

promiscuity.16

15

CONCLUSION

16

In summary, we have demonstrated that I152 in MiCGTb was the critical amino acid residue

17

for the second C-glycosylation and S60/V100/T104 were the key residues for bis-C-

18

glycosylation activity. Based on these determinant residues, we constructed a panel of evolved

19

CGTs with acceptor and donor promiscuity and enhanced proficiency. This work provides the

20

first example of probing and engineering key residues of CGTs for bis-C-glycosylation.

21

Moreover, given the expanded donor-acceptor substrate permissiveness of CGT variants, these

22

broadly promiscuous ‘universal’ enzymes hold promises to produce structurally diverse and

23

pharmacologically active bis-C-glycosyl derivatives with two identical or different sugar


16

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1

residues in drug discovery and will enhance ongoing efforts to develop combinatorial

2

biosynthesis and synthetic biology approaches for C-glycorandomization. We hope that the

3

advance described herein on the elucidation of structure–function relationships of plant CGT for

4

bis-C-glycosylation provides more practical implications for future engineering efforts to

5

produce novel CGTs with a versatile catalytic repertoire through design of the active sites of

6

CGTs.

7

EXPERIMENTAL SECTION

8

Chemicals and general methods. Chemical reagents were purchased from Sigma Aldrich (St.

9

Louis, MO, USA), J&K Scientific Ltd. (Beijing, China), and InnoChem Science & Technology

10

Co., Ltd. (Beijing, China). KOD-Plus DNA Polymerase was purchased from TOYOBO

11

BIOTECH Co., Ltd. (Shanghai, China). Primers synthesis and DNA sequencing were conducted

12

at Tsingke Biotech Company (Beijing, China). Restriction enzymes and DNA ligase were

13

purchased from Takara Biotechnology Co. Ltd. (Dalian, China). Enzymatic products were

14

detected using an Agilent 1200 series HPLC system (Agilent Technologies, Germany) coupled

15

with an LCQ Fleet ion trap mass spectrometer (Thermo Electron Corp., USA) equipped with an

16

electrospray ionization (ESI) source. The HRESIMS spectrum was performed using an Agilent

17

Technology 6520 Accurate Mass Q-TOF LC/MS Spectrometer. The conversion rates of the

18

enzyme reactions were calculated from peak areas of glycosylated products and substrates as

19

analyzed by HPLC at their maximum absorption wavelength, respectively. To facilitate

20

inferential statistical analysis, three parallel assays were routinely performed; the means±SD

21

from triplicate analyses are reported here. Compounds were characterized by 1H NMR at 400,

22

500 or 600 MHz and by

13

C NMR at 125 or 150 MHz on Mercury-400 and Bruker AVIIIHD


17

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Page 18 of 34

1

spectrometers. Chemical shifts (δ) were referenced to internal solvent resonances and were given

2

in parts per million (ppm). Coupling constants (J) were given in hertz (Hz).

3

Secondary structure prediction of MiCGTb and MiCGT. The deduced amino acid

4

sequences of MiCGTb and MiCGT were aligned with DNAMAN. The secondary structures of

5

MiCGTb

6

(http://www.compbio.dundee.ac.uk/jpred/).62 To compare their secondary structural differences,

7

the structural alignment of MiCGTb against MiCGT was constructed manually.

and

MiCGT

were

predicted

using

the

JPred4

program

8

Construction of MiCGTb-MiCGT chimeric genes. Ten chimeras were constructed by

9

swapping the targeted residues in protein regions A–E between MiCGT and MiCGTb, and the

10

corresponding chimeric genes were amplified by PCR using pET28a-MiCGTb or MiCGT as

11

templates and primers listed in Table S1. The PCR products were then purified by agarose gel

12

electrophoresis and transformed into Trans1-T1 Escherichia coli (TransGen Biotech, China).

13

The sequence of chimeric genes in the resulting plasmid (pET28a) was confirmed by Sanger

14

sequencing using the oligo-nucleotide primers T7 and T7-term.

15

Site-saturation mutagenesis of MiCGT or MiCGTb. Site-saturation mutagenesis of MiCGT

16

at site 152 and MiCGTb at the 60, 100 or 104 positions was performed by PCR using pET28a-

17

MiCGT and MiCGTb as templates, respectively, and the corresponding degenerate primers are

18

listed in Table S1. The PCR products were then purified by agarose gel electrophoresis and

19

transformed into Trans1-T1 E. coli. The sequence of mutant genes in the resulting plasmid

20

(pET28a) was confirmed by Sanger sequencing using the oligo-nucleotide primers T7 and T7-

21

term.

22

Construction of quadruple mutants of MiCGTb. Eight quadruple mutants of MiCGTb were

23

generated by combining the optimal residues for positions 60, 100, 104 and 152 together. The


18

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ACS Catalysis

1

corresponding amino acid in each mutant is listed in Table S3. The PCR products were then

2

purified by agarose gel electrophoresis and were transformed into Trans1-T1 E. coli. The

3

sequence of mutant genes in the resulting plasmid (pET28a) was confirmed by Sanger

4

sequencing using the oligo-nucleotide primers T7 and T7-term.

5

Expression and purification of mutant proteins. After the verification of the mutant

6

sequences, the recombinant plasmids were transformed into Transetta (DE3) E. coli for

7

heterologous expression. The proteins were induced and purified as described in our previous

8

work.18,19

9

HPLC-MS2-based bis-C-glycosylation activity assay. The reaction mixture containing 0.4

10

mM UDP-Glc (16), 0.2 mM acceptor (1–15) or mono-C-glycosides (1b–1h) and 100 µg of

11

purified protein in a final volume of 100 µL was incubated at pH 9.0 and 30 °C for 12 h. For

12

quantification, three parallel assays were routinely carried out. The reactions were terminated by

13

the addition of 200 µL of ice-cold MeOH and were centrifuged at 15,000 g for 30 min. The

14

supernatants were performed on a Merck RP-18 column (250 mm × 4.6 mm I.D., 5 µm, Merck

15

Co., Ltd., Germany) at a flow rate of 1 mL/min at 30 °C. The mobile phase was a gradient

16

elution of solvents A (MeOH) and B (0.1% formic acid aqueous solution). The gradient

17

programs were 30%–100% A, 25 min and 100% A, 5 min.

18

Effect of pH on bis-C-glycosylation activity. Enzymatic reactions were performed in various

19

reaction buffers with pH values in the range of 4.0–6.0 (citric acid-sodium citrate buffer), 6.0–

20

8.0 (Na2HPO4-NaH2PO4 buffer), 7.0–9.0 (Tris-HCl buffer) and 9.0–11.0 (Na2CO3-NaHCO3

21

buffer). All determinations were performed with UDP-Glc (16, 0.4 mM) as a donor and 2-

22

phenyl-2',4',6'-trihydroxyacetophenone (1, 0.2 mM) as an acceptor at 30 °C for 12 h. Three

23

parallel assays were routinely carried out. Aliquots were quenched with 200 µL of ice-cold


19

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Page 20 of 34

1

MeOH and were centrifuged at 15,000 g for 30 min. Supernatants were analyzed by analytical

2

reverse-phase HPLC as described above.

3

Determination of kinetic parameters. For the kinetic studies of sugar acceptors of CGT

4

variants, a typical assay containing 50 mM Tris-HCl (pH 9.0), saturating UDP-Glc (16, 800 µM)

5

and varying concentrations of 1 (10–300 µM) or 1b (10–300 µM) was conducted at pH 9.0 and

6

30 °C in a total volume of 100 µL. To determine the kinetic values of sugar donors, reactions

7

were performed with sugar donors (16, 18, 19, 21 and 23) from 50 to 400 µM with the saturating

8

1 (800 µM) at pH 9.0 and 30 °C in a total volume of 100 µL. Aliquots were quenched with 200

9

µL of ice-cold MeOH and were centrifuged at 15,000 g for 30 min. Supernatants were analyzed

10

by analytical reverse-phase HPLC as described above. All experiments were performed in

11

triplicate. The values of Km and kcat were calculated using the Lineweaver-Burk plot with

12

Origin8.1 software (Tables S4 and S5).

13

Tolerance of MiCGTb mutants for NDP-sugars. The sugar donors included UDP-α-D-

14

glucose (16), TDP-α-D-glucose (17), UDP-α-D-galactose (18), UDP-β-L-rhamnose (19), UDP-

15

β-L-arabinose (20), UDP-α-D-N-acetyl-glucosamine (21), UDP-α-D-xylose (22), UDP-α-D-

16

glucuronic acid (23), UDP-β-L-fucose (24), UDP-α-D-N-acetyl-galactosamine (25), GDP-α-D-

17

mannose (26) and UDP-α-D-N-acetyl-gulosamine (27). The sugar donors 19, 20 and 27 were

18

each prepared according to the literature.63–65 The sugar donors 22 and 24 were generated in one-

19

pot reactions coupled with OleD Loki.51 Other sugar donors were purchased from Sigma

20

Aldrich. All reactions containing 0.4 mM NDP-sugars, 0.2 mM 1 and 100 µg of purified protein

21

in a final volume of 100 µL were incubated at pH 9.0 and 30 °C for 12 h. Aliquots were

22

withdrawn, stopped by MeOH addition and analyzed by HPLC-MS2 as described above.


20

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ACS Catalysis

1

Preparation and structural identification of C-glycosyl products. The C-glycosylated

2

products were scale-up prepared with purified proteins at pH 9.0 and 30 °C for 12 h. The detailed

3

enzymatic procedure of each product was as follows. Bis-C-glucosides (1a–3a, 2c) and bis-C-

4

alkylation products (15a and 15b) were scale-up prepared with acceptor/UDP-Glc (mol/mol 1/2)

5

and 10 mg purified proteins MiCGTb in a final volume of 10 mL. Mono-C-glycosides (1c–1h)

6

were scale-up prepared with acceptor/NDP-sugar (mol/mol 1/2) and 10 mg purified proteins

7

MiCGTb-GAGM in a final volume of 10 mL. Bis-C-glycosides (1ca, 1da and 1ga) were scale-

8

up prepared with 1 and UDP-sugar (mol/mol 1/2) and 10 mg purified proteins MiCGTb-GAGM

9

in a final volume of 10 mL; after 12 h’s incubation, the same mole of UDP-Glc was added into

10

the mixtures for another 12 h. All reactions were monitored by HPLC. The reaction mixtures

11

were quenched by adding 40 mL of ethyl acetate and extracted 5 times. The organic solvent was

12

evaporated under reduced pressure; the afforded residue was dissolved in 1.5 mL of MeOH and

13

purified by reverse-phase semi-preparative HPLC with YMC-pack ODS-A (250 mm×10 mm,

14

I.D., 5 µm, YMC, Japan) at a flow rate of 3 mL/ min at 30 °C. The mobile phase was a gradient

15

elution of solvents A (MeOH) and B (H2O). The gradient programs were 30%–70% A, 20 min,

16

70%–100% A, 5 min and 100% A, 5 min. The obtained products were solved in MeOH-d4 and

17

analyzed by MS, 1H NMR and 13C NMR (Figures S35–S66 in the Supporting Information).

18

Construction of a binary complex. To build a 3D structure of the variants bound with mono-

19

C-glucoside 1b, the structural modeling of MiCGT, MiCGT-E152I, MiCGTb and MiCGTb-

20

I152E were built using the program Modeller V9.13 based on the crystal structure of UGT71G1

21

(PDB code 2ACW),41 respectively. To evaluate the accuracy of the model, PROCHECK was

22

employed.66 Docking simulations between the enzymes and 1b were undertaken in AutoDock

23

Vina.67


21

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Page 22 of 34

1

Molecular dynamics simulations. Molecular dynamics (MD) simulations of the MiCGT–1b,

2

MiCGT-E152I–1b, and MiCGTb–1b, MiCGTb-I152E–1b complex were performed using

3

Amber12 software package. The amber ff12SB force field was applied for the protein. Force

4

field parameters of ligand 1b was generated using the antechamber module of AMBER 12 and

5

the general AMBER force field (GAFF).68 TIP3P water molecules were utilized to solvate the

6

complex, extending at least 10 Å from the protein.69 Six Na+ ions were added for charge

7

neutralization. To remove the bad contacts, the system was subjected to energy minimization.

8

First, the water molecules and ions were refined through 2,500 steps of the steepest descent

9

followed by 2,500 steps of the conjugate gradient, keeping the protein and ligands fixed. Second,

10

the whole system was relaxed by 10,000 cycles of the minimization procedure with 5,000 cycles

11

of the steepest descent and 5,000 cycles of the conjugate gradient minimization. Thereafter, the

12

system was heated from 0 to 310 K by 500 ps position restraint simulation. A 2-ns MD

13

simulations without any restraints were sequentially performed to equilibrate the complex.

14

Finally, a length of a 20-ns trajectory was computed at 310 K under constant pressure, and

15

production MD simulations were carried out utilizing the GPU accelerated pmemd.cuda code.

16

All of the MD results were analyzed using the ptraj module of the Amber 12 software package.

17

Docking simulations among the MiCGT–1b, MiCGT-E152I–1b, MiCGTb–1b and MiCGTb-

18

I152E–1b complex and UDP-Glc (16) were undertaken in AutoDock Vina.67

19

Circular dichroism and fluorescence spectroscopy. Circular dichroism (CD) experiments

20

were performed using a Chirascan-plus spectrometer (Applied Photophysics, UK) in a step-scan

21

mode averaging over three runs using quartz cuvette with a 1-mm path length.16 The spectra

22

were recorded in the wavelength ranges from 190 to 260 nm and in ∆A (M−1 cm−1) for residue in

23

the function of path length λ (nm) at 25 °C. Fluorescence measurements were acquired using a


22

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ACS Catalysis

1

Dual-FL spectrometer (Horiba, Japan) and 0.1 mL of samples in a 1-cm path length of quartz

2

cells.16 A bandwidth of 5 nm was used for the excitation and emission beams. The excitation

3

wavelength was fixed at 270 nm, and the emission spectra were recorded at 25 °C from 290–400

4

nm at a scan rate of 100 nm/min. All samples were prepared in a buffer containing 20 mM

5

NaH2PO4-Na2HPO4 (pH 8.0) with a final protein concentration maintained at 0.1 mg/ml.

6 7

AUTHOR INFORMATION

8

Corresponding Author

9

* E-mail: [email protected]

10

Notes

11

The authors declare no competing financial interest.

12

ASSOCIATED CONTENT

13

Supporting Information

14

Bis-C-glycosylation activity of CGT variants, biochemical characteristics of CGT variants,

15

HPLC-MS, HR-ESI-MS and NMR characterization data and spectra of C-glycosylated products,

16

MD simulations, CD and fluorescence spectra (PDF). This material is available free of charge

17

via the Internet at http://pubs.acs.org.

18

ACKNOWLEDGMENT

19

We thank Prof. Yihua Chen (Institute of Microbiology, Chinese Academy of Sciences, China)

20

for providing the sugar donor (UDP-α-D-N-acetyl-gulosamine). This work was supported by the


23

ACS Catalysis 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

Page 24 of 34

1

National Natural Science Foundation of China (Grant Nos. 81703369, 21572277, and 81573317)

2

and CAMS Innovation Fund for Medical Sciences (CIFMS-2016-I2M-3-012).

3

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4 5 6 7 8 9

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Roots of Desmodium incanum (G. Mey.) DC. Org. Biomol. Chem. 2015, 13, 11663−11673.

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Recombinant Oryza sativa C-Glycosyltransferase (OsCGT) and Desmodium incanum Root

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Luzhetskyy, A.; Bechthold, A. Rational Design of an Aryl-C-Glycoside Catalyst from a Natural

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