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. 2022 Feb 2;7(1):602–620. doi: 10.1016/j.synbio.2022.01.001

Table 2.

The structure-based rational design and directed evolution of GTs that involved in the biosynthesis of glycosylated plant natural products.

Protein name Organism Genbank PDB code Representative schematic reaction Key residues Engineering Reference
YjiC Bacillus subtilis NP_389104.1 7BOV Image 1
  • ●

    Ser277 is critical for Nucleoside Diphosphate (NDP) recognition

  • ●

    Glu317, Gln318, Ser128 and Ser129 are crucial for glycosyl moiety recognition
  • ●

    V108A increase 5-fold for UDP glycosylation activity and improve 35% for pterostilbene glycosylation under the existence UDPG.

  • ●

    L320A improve 2-fold for Kcat/KM while add 65% activity for ADPG.

 [129]
UGT72B1 Arabidopsis thaliana CAB80916.1 2VCE Image 2
  • ●

    His19 is positioned to act as a Brønsted base

  • ●

    Gln389 and Glu388 interact with the glucose moiety of donors

  • ●

    Glu83, Ile86, Leu118, Phe119, Phe148, Leu183, and Leu197 are predominant in the acceptor binding

 – [130]
UGT74F2 Arabidopsis thaliana AAB64024.1 5U6M Image 3
  • ●

    His18 shows a central role in catalysis

  • ●

    Tyr180 is important for ligand recognition or binding

  • ●

    Met274 could be crucial for orientation of the salicylic acid

 – [131]
UGT89C1 Arabidopsis thaliana AAF80123.1 6IJ7 Image 4
  • ●

    Asp356, His357, Pro147 and Ile148 are key residues for sugar donor recognition and specificity for UDP-β-l-rhamnose.

  • ●

    His21 is a key residue as the catalytic base and the only catalytic residue involved in catalysis.
  • ●

    H357Q exhibited activity with both UDP-β-l-rhamnose and UDP-glucose

 [132]
UGT78K6 Clitoria ternatea BAF49297.1 3WC4 Image 5
  • ●

    Pro78, Asp181 and Asp367 are involved in the acceptor binding.

  • ●

    Asn137 could participate in the recognition of the glucose moiety.

  • ●

    His17 is the key catalytic residue.

 – [133,134]
UGT708C1 Fagopyrum esculentum BAP90360.1 6LLG Image 6
  • ●

    Asp382, Gln383, Thr151 and Thr150 play important role in the recognition of sugar moiety.

  • ●

    Phe130, Tyr102 and Phe198 bound and stabilize the acceptor.

  • ●

    Arg280 and Asp96 play important roles in the catalytic activity.

 – [135]
GgCGT Glycyrrhiza glabra QGL05036.1 6L5P Image 7
  • ●

    R285, T145, D390 and Q391 determine the sugar donor preference.

  • ●

    The flopropione unit is the minimum required unit for the di-C-glycosylation due to the interactions of its 2’-/6′-OH with H27.

  • ●

    The spacious substrate-binding tunnel near G389 is critical for the di-C-glycosylation activity and the broad substrate promiscuity.
  • ●

    G389K mutation could switch di- to mono-C-glycosylation

 [48]
UGT73P12 Glycyrrhiza uralensis BBN60799.1 7C2X Image 8
  • ●

    Arg32 is the essential residue to provide high specificity for UDP-glucuronic acid.

 – [136]
LpCGTa Landoltia punctata QLF98869.1 6LG1 Image 9 – – [76]
LpCGTb Landoltia punctata QLF98870.1 6LFN Image 10 – – [76]
SbCGTa Scutellaria baicalensis QLF98861.1 6LG0 Image 11
  • ●

    His24 is critical to initiate the catalytic reaction through deprotonation of the substrate.

 – [76]
SbCGTb Scutellaria baicalensi QLF98862.1 6LFZ Image 12
  • ●

    His23 is critical to initiate the catalytic reaction through deprotonation of the substrate.
  • ●

    R94 M/I143 M/V144T/T145S/H194D/G275T/P374Q mutant had switched the function of SbCGTb to SbCGTa

 [76]
UGT708A6 Zea mays ACF81582.1 6LF6 Image 13 – – [76]
UGT71G1 Medicago truncatula AAW56092.1 2ACV Image 14
  • ●

    His22 is the catalytic base.

  • ●

    Asp121 is a key residue that may assist deprotonation of the acceptor.

  • ●

    Glu381 is the key residue in recognition of the sugar donor.

 – [2]
UGT85H2 Medicago truncatula ABE87250.1 2PQ6 Image 15
  • ●

    His21 and Asp125 are essential for catalytic activity.

 – [137]
UGT78G1 Medicago truncatula ABI94025.1 3HBF Image 16
  • ●

    Glu192 is the key residue for the reverse reaction.

  • ●

    His26 act as the catalytic residue.

  • ●

    Asp124 also plays an essential role in catalysis.

 – [138]
Os79 Oryza sativa BAF14158.1 5TMB Image 17
  • ●

    His27 activate the trichothecene O3 hydroxyl for nucleophilic attack at C1’ of the UDP-glucose donor.

  • ●

    Thr291 plays a critical role in catalysis as a catalytic acid or to position the UDP moiety during the nucleophilic attack.

 – [139]
PtUGT1 Persicaria tinctoria BBB06426.1 5NLM Image 18
  • ●

    E88 could play a major role in indoxyl specificity and turnover.

  • ●

    H26 is expected to be the Brønsted base.

  • ●

    D122 is believed to balance the charge on the catalytic histidine.

 – [140]
PaGT2 Phytolacca americana BAG71125.1 6JEL Image 19
  • ●

    His18 and His81 are recognized as the catalytic residues.
  • ●

    C142A and C142F mutants formed resveratrol 3-O-β-glucoside and resveratrol 4′-O-β-glucoside, respectively, with high regioselectivity.

 [42]
PaGT3 Phytolacca americana BAG71127.1 6LZX Image 20
  • ●

    His20 is the active-site residue.

  • ●

    Trp417 and Arg419 are actively participate in the formation of the acceptor-binding pocket.

 – [141]
UGT51 Saccharomyces cerevisiae AAB67475.1 5GL5 Image 21
  • ●

    Asp752 serve as a catalytic base.

  • ●

    Met851 is important for UGT51 activity.

  • ●

    Gln1094, Asp1093 and Ser1072 make several critical interactions with the glucose moiety of donor.
  • ●

    A mutant M7_1 (S81A/L82A/V84A/K92A/E96K/S129A/N172D) presented an ∼1800-fold activity improvement toward an unnatural substrate protopanaxadiol.

 [142,143]
UGT74AC1 Siraitia grosvenorii AEM42999.1 6L8W Image 22
  • ●

    His18 is the general base abstracts a proton from the 3-hydroxyl group of mogrol.

  • ●

    Asp111 stabilizes the catalytic conformation and balance the charge.
  • ●

    Mutant M4 (T79Y/R28H/L48 M/L109I) showed ∼200-fold higher activity than WT.

  • ●

    Mutant M5 (T79Y/R28H/L48 M/L109I/S15A) showed ∼3.8-fold higher than M4.

 [144]
UGT74AC2 Siraitia grosvenorii AXK92493.1 7BV3 Image 23
  • ●

    The uracil ring forms hydrogen bonds and parallel π-stacking interactions with A353 and W352, respectively, and the ribose ring interacts with the enzyme through hydrogen bonds with E378 and Q355, while the α-phosphate forms hydrogen bonds with H370, N374 and S375

  • ●

    The acceptor binding pocket is constructed by 13 residues, where P12, L43 and V91 are located at the entrance, and V190, M196 as well as L200 are situated at the bottom
  • ●

    Mutant G11Y is found that shows 75% selectivity and >99% conversion towards silybin A-3,7-O-diglucoside.

  • ●

    Three variants show enhanced regioselectivity toward silybin A-7-O-glucoside, P12Y (81% selectivity and 68% conversion), L200W (92% selectivity and 61% conversion) and Y145W (89% selectivity and 75% conversion)

 [145]
UGT76G1 Stevia rebaudiana AAR06912.1 6INF Image 24
  • ●

    His25 is the general base, which deprotonates the 3-hydroxyl of the accepting glucose A to activate it as a nucleophile.

  • ●

    Asp124 plays an important catalytic role in relaying protons off and on His25.

 – [146]
TcCGT1 Trollius chinensis QCZ42162.1 6JTD Image 25
  • ●

    H24 acts to stabilize both the deprotonated substrate and the product sugar, though it is not indispensable for the glycosylation activity.

  • ●

    E396 plays an important role to stabilize and orient the UDP–Glc sugar.
  • ●

    I94E and G284K switch C- to O-glycosylation.

 [49]
VvGT1 Vitis vinifera AAB81683.1 2C1X Image 26
  • ●

    Asp374, Gln375 and Thr141 are key players in sugar recognition.

 – [147]
UGTPg45 Panax ginseng AKA44586.1 – Image 27 –
  • ●

    A mutant UGTPg45-HV with two missense mutations (Q222H and A322V) gave 70% increase of ginsenoside Rh2 yield

 [148]