Article Figures & Data
Figures
- FIG 1
Reactions of GDH and APRT. (A) The GDH reaction. (B) The APRT reaction.
- FIG 2
Isolation of the partner protein of TtGDH. (A) Pulldown assay for His-tagged GdhA using a Ni2+-NTA column. Lane M, molecular size markers; lane S, supernatant of cell extract of the Tt27NStHisAPRTh strain; lane F, flowthrough fraction from the column; lane W, wash fraction from the column; lane E, elution fraction from the column. (B) Copurification of Strep-tagged ARPTh with TtGDH. The 50-fold concentrated elution fraction from the Strep-Tactin column was applied. Lane 1, Tt27NStHisAPRTh strain; lane 2, wild-type T. thermophilus HB27 strain.
- FIG 3
Amino acid sequence alignment of APRTh and APRT. Tt27APRTh, APRTh from T. thermophilus HB27; TO73_0477, APRTh from T. aquaticus; Ocepr_0780, APRTh from Oceanithermus profundus; Deide13870, APRTh from Deinococcus deserti; SU48_05320, APRTh from Deinococcus puniceus. Tt27APRT, APRT from T. thermophilus HB27; TO73_0476, APRT from T. aquaticus; Ocepr_0779, APRT from Oceanithermus profundus; Deide13880, APRT from Deinococcus deserti; SU48_05315, APRT from Deinococcus puniceus. ScAPRT, APRT from S. cerevisiae. Black boxes at the bottom indicate the residues demonstrated to play important roles in the catalytic reaction of ScAPRT. The corresponding residues in the related proteins are boxed with bold lines.
- FIG 4
Coexpression and purification of TtGDH. (A) Copurification of APRTh with a His tag at the N terminus, GdhA, and GdhB. (B) Gel filtration column chromatography of proteins copurified with His-tagged APRTh. Chromatogram of absorption at 280 nm. AU, arbitrary units. The estimated molecular weights of the peaks are shown. (C) SDS-PAGE of the collected fractions from chromatography. The numbers of corresponding elution volumes are indicated for several fractions.
- FIG 5
Copurification of APRTh with a His tag at the N terminus and Gdh subunits. (A) Copurification of APRTh with a His tag at the N terminus and GdhB. (B) Copurification of APRTh with a His tag at the N terminus and GdhA. Lane M, molecular size markers; lane S, supernatant of cell extract of the strain; lane F, flowthrough fraction from the column; lane W, wash fraction from the column; lane E, elution fraction from the column.
- FIG 6
Activation profiles of GDH activity of the TtGDH-APRTh complex by leucine (A) and AMP (B). The concentrations of supplemented effectors are shown at the bottom. The gray and white bars indicate the reductive amination and oxidative deamination reactions, respectively.
- FIG 7
Growth curves of T. thermophilus and the recombinant strains in minimal medium. WT, wild-type strain; Tt27ΔAPRTh, aprth knockout strain; Tt27NStHisAPRTh, aprth overexpression strains. O.D.600, optical density at 600 nm.
Tables
- TABLE 1
APRT activity of TTC1250 (APRT) and TTC1249 (APRTh)
Substrate APRT activity (U/mg) of: TTC1250 (APRT) TTC1249 (APRTh) Adenine 27 ± 0.17 <1 Guanine <1 <1 Hypoxanthine <1 <1 Xanthine <1 <1 - TABLE 2
Effects of metabolic compounds on GDH activity
Compound TtGDH APRTha TtGDHb Reductive amination (%) Oxidative deamination (%) Reductive amination (%) Oxidative deamination (%) None 100 ± 2 100 ± 1 100 ± 4 100 ± 1 AMP 626 ± 19 252 ± 1 98 ± 2 104 ± 3 ADP 152 ± 6 120 ± 1 ND ND ATP 124 ± 5 111 ± 5 ND ND GMP 133 ± 6 101 ± 1 ND ND GDP 113 ± 9 103 ± 1 ND ND GTP 108 ± 15 105 ± 6 ND ND IMP 116 ± 5 100 ± 1 ND ND Leu 2,440 ± 134 335 ± 3 459 ± 14 432 ± 2 Leu/AMP 5,670 ± 102 457 ± 6 470 ± 14 426 ± 1 - TABLE 3
Kinetic parameters of TtGDH-APRTh
Substrate Without effector AMP Leu AMP/Leu Kmapp (μM) kcatapp (s−1) Kmapp (μM) kcatapp (s−1) Kmapp (μM) kcatapp (s−1) Kmapp (μM) kcatapp (s−1) Reductive amination NADH 8.0 ± 2.4 0.20 ± 0.017 5.8 ± 1.9 0.72 ± 0.05 3.0 ± 0.7 9.1 ± 0.3 5.3 ± 1.0 29 ± 1.0 2-OG 5.1 ± 0.7 0.23 ± 0.006 22.9 ± 1.1 0.66 ± 0.009 27 ± 0.1 15 ± 1.7 52 ± 1.5 37 ± 0.4 NH4+ 29,000 ± 10,000 0.31 ± 0.054 43,000 ± 11,000 1.1 ± 0.15 46,000 ± 7,900 16 ± 1.6 33,000 ± 1,600 44 ± 1.0 Oxidative deamination NAD+ 24 ± 0.95 2.8 ± 0.05 130 ± 22 9.1 ± 0.4 56 ± 5.2 3.7 ± 0.09 160 ± 12 12 ± 0.20 Glu 450 ± 26 4.5 ± 0.04 770 ± 57 13 ± 0.24 Glu (high [Glu]) 52,000 ± 15,000 1.5 ± 0.1 19,0000 ± 55,000 4.4 ± 1.0 Glu (low [Glu]) 1,200 ± 82 0.27 ± 0.01 270 ± 50 1.2 ± 0.1
Supplemental material
- Supplemental file 1 -
Fig. S1 (Construction of plasmids), S2 to S4 (Gel filtration column chromatography), S5 (Interaction between TtGDH and APRTh), and S6 (Tandem coordination of gdh and aprt in several organisms) and Tables S1 and S2 (Oligonucleotides)
PDF, 586K
- Supplemental file 1 -
