Article Figures & Data
Figures
- Figure 1.
HPLC elution profiles for diCQA production by HCT. A, Native HCT reaction activity. B, His-154-Asn mutant HCT reaction activity. Enzyme at 0.5 µm was incubated with 1 mm 5-CQA and 1 mm CoA, and samples were taken at 15 min (top panels) and after overnight incubation (O/N; bottom panels). The relevant control reactions are shown in Supplemental Figure S8.
- Figure 2.
The overall structures of HCT and TRI101 are composed of two structurally related domains. A, The HCT N-terminal domain (residues 1–189) is colored in blue and the C-terminal domain (residues 224–434) is colored in gold. B, TRI101 in complex with CoA and DON. The N- and C-terminal domains are colored as for HCT, and the TRI101 substrates are shown in gray (CoA) or green (DON) balls.
- Figure 3.
Comparison of the native and Lys-HCT crystal structures with the TRI101-DON complex in the active site region. A, Native HCT molecule 1. B, Native HCT molecule 2 (similar to Lys mutant crystal form 2). C, Lys mutant crystal form 1. D, TRI101-DON complex structure. All the structures are shown in the same relative orientation to highlight the conformation of the Val-31 to Pro-37 loop in HCT. The His residues found in the active site region of HCT and TRI101 are show as sticks, and DON is colored in green.
- Figure 4.
Comparison of the best docking results obtained for HCT with the TRI101-DON complex. A, Quinic acid in SBP1. B, Caffeic acid in SBP2. C, 5-CQA encompassing SBP1 and -2. D, TRI101-DON complex. E, TRI101 precludes hydroxycinnamic acid binding. F, 3,5-diCQA showing SBP1 and SPB3. Residues lining the substrate-binding site are shown as sticks, with substrates colored in green. G, Sequence and structure alignment of HCT with other hydroxycinnamoyl transferases and representative members of the BAHD superfamily. Secondary structures of the native HCT and FsTRI101 are shown above and below the alignment, respectively. Residues predicted to be involved in substrate binding by HCT are denoted as gray circles for CoA, magenta polygons for hydroxycinnamic moiety, and green stars for acyl acceptor moiety. A full alignment is shown in Supplemental Figure S9.
Tables
- Table I. Data collection and refinement statistics
Values in parentheses are for the outermost resolution shell
calculated for the whole data set . Rfree was calculated as for Rcryst, with 5% of the data omitted from the structural refinement.Crystal Parameters Crystal Name HCT Lys-HCT Form 1 Lys-HCT Form 2 Data collection Wavelength (Å) 0.873 0.939 0.976 Space group P42212 P212121 C2221 Unit-cell dimensions (Å) a = 116.1 a = 63.55 a = 76.55 b = 116.1 b = 116.48 b = 96.28 c = 158.9 c = 118.04 c = 118.76 Molecules 2 2 1 Resolution range (Å) 45–3.0 (3.2–3.0) 50–1.7 (1.8–1.7) 50–2.5 (2.6–2.5) Observed reflections [I/σ(I)>0] 72,116 674,662 55,469 Unique reflections 22,073 92,179 14,082 Completeness (%) 99.0 (98.4) 95.0 (83.9) 90.5 (79.2) Rmerge (%) 16.6 (56.6) 9.6 (53.3) 7.1 (42.7) <I/σ(I)> 8.5 (3.1) 12.7 (2.8) 16.8 (3.5) Model quality indicators Rcryst (%) 0.189 0.168 0.194 Rfree (%) 0.250 0.203 0.254 Mean B-factor (Å2) 11.6 19.8 34.0 Root-mean-square-deviation bonds (Å)/angles (°) 0.011/1.5 0.026/2.0 0.012/1.5 - Table II. Steady-state kinetic parameters of native and mutant HCT
Assays were performed as described in “Materials and Methods,” and average values ± se (n = 3) are also shown.
HCT Shikimate Quinate 5-CQA Km Vmax Vmax/Km Km Vmax Vmax/Km Km Vmax Vmax/Km μm nkat mg−1 μm nkat mg−1 μm nkat mg−1 Native 75.0 ± 8.5 13.55 ± 0.38 180,700 430 ± 112 0.74 ± 0.10 1,720 757 ± 100 0.072 ± 0.003 95.1 Lys-HCT 30.7 ± 6.4 7.92 ± 0.34 258,000 608 ± 93 0.070 ± 0.004 115 His-154-Asn 35.4 ± 7.1 6.83 ± 0.44 192,900 351 ± 47 0.635 ± 0.025 1,809 Leu-400-Thr 640 ± 91 7.64 ± 0.47 11,940 162 ± 14 6.22 ± 0.20 38,400 1,930 ± 610 0.063 ± 0.010 32.6 Phe-402-Tyr 316 ± 31 11.32 ± 0.38 35,820 233 ± 20 3.73 ± 0.14 16,010 745 ± 90 0.223 ± 0.010 299 Leu-400-Thr/Phe-402-Tyr 1,160 ± 191 3.64 ± 0.25 3,137 99 ± 6 7.98 ± 0.15 80,610 210 ± 28 0.161 ± 0.005 767
Supplemental Data
Supplemental Figures
Files in this Data Supplement:
- Supplemental Data - Supplemental Figures 1-14
- Supplemental Data - Supplemental Figure Legends
