Fig 1.
Synthesis pathway of tetrapyrroles.
ALA, 5-aminolevulinic acid; PBGS, porphobilinogen synthase; PBGD, porphobilinogen deaminase; UROS, uroporphyrinogen III synthase; SUMT, S-adenosyl-l-methionine-dependent urogen III methyltransferase; SAM, S-adenosyl-l-methionine; SAH, S-adenosylhomocysteine
Fig 2.
UV-visible absorption spectra of products in the tandem enzyme assay.
Precorrin-2 was produced from ALA by the tandem-enzyme reaction system containing purified, recombinant PBGS, PBGD, UROS, SUMT, and SAM (dotted line); precorrin-2 was then converted into sirohydrochlorin by precorrin-2 dehydrogenase in the presence of NAD (solid line).
Fig 3.
Titration of substrates and cofactors.
(A), Optimization of SAM concentration. The reaction mixture contained: 1 mM ALA, 200 μM NAD, 1 μM each enzyme, and various concentrations of SAM (20 μM, 50 μM, 200 μM, 500 μM, and 2 mM SAM); (B), Optimization of ALA concentration. The reaction mixture contained: 200 μM SAM, 200 μM NAD, 1 μM each enzyme, and various concentrations of ALA (0.5 mM, 1 mM, 5 mM, 20 mM, and 100 mM). Results are presented as mean ± SD. Error bars represent standard deviations of three biological replicates.
Fig 4.
Titration of each enzyme involved in precorrin-2 synthesis.
The reaction mixture contained: 5 mM ALA, 200 μM SAM, 1 mM NAD, 1 μM precorrin-2 dehydrogenase, and various concentrations of titrated enzymes. (A), Optimization of PBGS concentration. The reaction mixture contained 1 μM PBGD, 1 μM UROS, 1 μM SUMT, and various concentrations of PBGS from 0.02–3 μM. (B), Optimization of PBGD concentration. The reaction mixture contained 0.1 μM PBGS, 1 μM UROS, 1 μM SUMT, and various concentrations of PBGD from 0.1–6 μM. (C), Optimization of UROS concentration. The reaction mixture contained 0.1 μM PBGS, 1 μM PBGD, 1 μM SUMT, and various concentrations of UROS from 0.1–10 μM. (D), Optimization of SUMT concentration. The reaction mixture contained 0.1 μM PBGS, 1 μM PBGD, 1 μM UROS, and various concentrations of SUMT from 0.1–35 μM. Results are presented as the mean of 3 replicates. Error bars indicate SD.
Table 1.
Box-Behnken design (BBD) with the experimental responses.
Fig 5.
Surface response plots showing the effects of varying PBGS, PBGD, UROS, and SUMT concentrations.
(A), Effect of PBGS and PBGD concentrations. (B), Effect of PBGD and UROS concentrations. (C), Effect of UROS and SUMT concentrations. (D), Effect of PBGS and SUMT concentrations.
Table 2.
Analysis of variance (ANOVA) for response surface quadratic model.
Table 3.
Analysis of variance (ANOVA) for reduced response surface quadratic model.