Enhancing thermostability and removing hemin inhibition of Rhodopseudomonas palustris 5-aminolevulinic acid synthase by computer-aided rational design
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To enhance the thermostability and deregulate the hemin inhibition of 5-aminolevulinic acid (ALA) synthase from Rhodopseudomonas palustris (RP-ALAS) by a computer-aided rational design strategy.
Eighteen RP-ALAS single variants were rationally designed and screened by measuring their residual activities upon heating. Among them, H29R and H15K exhibited a 2.3 °C and 6.0 °C higher melting temperature than wild-type, respectively. A 6.7-fold and 10.3-fold increase in specific activity after 1 h incubation at 37 °C was obtained for H29R (2.0 U/mg) and H15K (3.1 U/mg) compared to wild-type (0.3 U/mg). Additionally, higher residual activities in the presence of hemin were obtained for H29R and H15K (e.g., 64% and 76% at 10 μM hemin vs. 27% for wild-type). The ALA titer was increased by 6% and 22% in fermentation using Corynebacterium glutamicum ATCC 13032 expressing H29R and H15K, respectively.
H29R and H15K showed high thermostability, reduced hemin inhibition and slightly high activity, indicating that these two variants are good candidates for bioproduction of ALA.
Keywords5-Aminolevulinic acid synthase Hemin inhibition Rational design Rhodopseudomonas palustris Thermal stability
We are grateful for the financial support from National Natural Science Foundation of China (No. 21606251), the Key Research Program of the Chinese Academy of Sciences (No. KFZD-SW-212), the Tianjin Municipal City, the first “Special Support Plan for Talents Development” and “High-level Innovation and Entrepreneurship Team”, and Science and Technology Project of Tianjin (Nos. 15PTCYSY00020 and 14ZCZDSY00058). We thank Taiwo Dele-Osibanjo (Tianjin Institute of Industrial Biotechnology) for critical reading and editing of the manuscript.
Supplementary Table 1—Primers used in this study.
Supplementary Table 2—The occurring frequency of amino acids at histidine positions. The data are shown as percentage values.
Supplementary Table 3—The values of the folding free energy change (∆∆G) predicted by FoldX. The unit is kcal/mol.
Supplementary Fig. 1—3D model of wild-type RP-ALAS. The homology model of wild-type RP-ALAS was constructed using the crystal structure of ALAS from R. capsulatus (PDB code: 2BWP) as template.
Supplementary Fig. 2—SDS-PAGE analysis of the purified ALASs. Lane M, protein marker; lane 1, wild-type; lane 2, H29R; lane 3, H15K.
Supplementary Fig. 3—Absorption spectra of hemin-RP-ALAS (black squares), hemin-H29R (green triangles) and hemin-H15K (red circles) complex. The concentrations of hemin were 20 and of ALAS were (a) 10 μM (874 μg/mL), (b) 4 μM (349.6 μg/mL) and (c) 2 μM (174.8 μg/mL), respectively.
Supplementary Fig. 4—ALA production using Corynebacterium glutamicum ATCC 13032 expressing wild-type, H29R and H15K.
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Conflict of interest
The authors declare that they have no conflict of interest.
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