8CEK

Succinyl-CoA Reductase from Clostridium kluyveri (SucD) with NADPH

8CEK の概要

• エントリーDOI: 10.2210/pdb8cek/pdb
• 分子名称: Succinate-semialdehyde dehydrogenase (acetylating), NADP NICOTINAMIDE-ADENINE-DINUCLEOTIDE PHOSPHATE (3 entities in total)
• 機能のキーワード: ssa, succinic semialdehyde, nadph, nadp+, sucd, ssr, succinyl-coa, mesaconyl-coa, mesaconyl-c1-coa, sucd_ck, cksucd, oxidoreductase
• 由来する生物種: Clostridium kluyveri
• タンパク質・核酸の鎖数: 4
• 化学式量合計: 199093.26

構造登録者

Pfister, P.,Diehl, C.,Erb, T.J. (登録日: 2023-02-02, 公開日: 2023-05-31, 最終更新日: 2023-09-20)

主引用文献

Pfister, P.,Diehl, C.,Hammarlund, E.,Carrillo, M.,Erb, T.J.
Enhancing the Substrate Specificity of Clostridium Succinyl-CoA Reductase for Synthetic Biology and Biocatalysis.
Biochemistry, 62:1786-1793, 2023

PubMed Abstract: Succinyl-CoA reductase (SucD) is an acylating aldehyde reductase that catalyzes the NADPH-dependent reduction of succinyl-CoA to succinic semialdehyde. The reaction sequence from succinate to crotonyl-CoA is of particular interest for several new-to-nature CO-fixation pathways, such as the crotonyl-CoA/ethylmalonyl-CoA/hydroxybutyryl-CoA (CETCH) cycle, in which SucD plays a key role. However, pathways like the CETCH cycle feature several CoA-ester intermediates, which could be potentially side substrates for this enzyme. Here, we show that the side reaction for most CETCH cycle metabolites is relatively small (<2%) with the exception of mesaconyl-C1-CoA (16%), which represents a competing substrate in this pathway. We addressed this promiscuity by solving the crystal structure of a SucD of in complex with NADP and mesaconyl-C1-CoA. We further identified two residues (Lys70 and Ser243) that coordinate mesaconyl-C1-CoA at the active site. We targeted those residues with site-directed mutagenesis to improve succinyl-CoA over mesaconyl-C1-CoA reduction. The best resulting SucD variant, K70R, showed a strongly reduced side activity for mesaconyl-C1-CoA, but the substitution also reduced the specific activity for succinyl-CoA by a factor of 10. Transferring the same mutations into a SucD homologue from similarly decreases the side reaction of this enzyme for mesaconyl-C1-CoA from 12 to 2%, notably without changing the catalytic efficiency for succinyl-CoA. Overall, our structure-based engineering efforts provided a highly specific enzyme of interest for several applications in biocatalysis and synthetic biology.

PubMed: 37207322
DOI: 10.1021/acs.biochem.3c00102

実験手法

X-RAY DIFFRACTION (2.15 Å)