7X1L

Malate dehydrogenase from Geobacillus stearothermophilus (gs-MDH) delta E311 mutant complexed with Nicotinamide Adenine Dinucleotide (NAD+)

Summary for 7X1L

• Entry DOI: 10.2210/pdb7x1l/pdb
• Descriptor: Malate dehydrogenase, NICOTINAMIDE-ADENINE-DINUCLEOTIDE (3 entities in total)
• Functional Keywords: dehydrogease, oxidoreductase
• Biological source: Geobacillus stearothermophilus
• Total number of polymer chains: 6
• Total formula weight: 218799.74

Authors

Shimozawa, Y.,Himiyama, T.,Nakamura, T.,Nishiya, Y. (deposition date: 2022-02-24, release date: 2022-10-19, Last modification date: 2023-11-29)

Primary citation

Shimozawa, Y.,Matsuhisa, H.,Nakamura, T.,Himiyama, T.,Nishiya, Y.
Reducing substrate inhibition of malate dehydrogenase from Geobacillus stearothermophilus by C-terminal truncation.
Protein Eng.Des.Sel., 35:-, 2022

PubMed Abstract: Malate dehydrogenase (MDH) catalyzes the reduction of oxaloacetate to L-malate. Geobacillus stearothermophilus MDH (gs-MDH) is used as a diagnostic reagent; however, gs-MDH is robustly inhibited at high substrate concentrations, which limits its reaction rate. Here, we reduced substrate inhibition of gs-MDH by deleting its C-terminal residues. Computational analysis showed that C-terminal residues regulate the position of the active site loop. C-terminal deletions of gs-MDH successfully increased Ki values by 5- to 8-fold with maintained thermal stability (>90% of the wild-type enzyme), although kcat/Km values were decreased by <2-fold. The structure of the mutant showed a shift in the location of the active site loop and a decrease in its volume, suggesting that substrate inhibition was reduced by eliminating the putative substrate binding site causing inhibition. Our results provide an effective method to reduce substrate inhibition of the enzyme without loss of other parameters, including binding and stability constants.

PubMed: 36208218
DOI: 10.1093/protein/gzac008

Experimental method

X-RAY DIFFRACTION (2.28 Å)