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Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering

Holmberg, Niklas; Ryde, U LU and Bülow, L LU (1999) In Protein Engineering 12(10). p.6-851
Abstract

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant,... (More)

L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.

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published
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keywords
Amino Acid Motifs, Binding Sites, Cloning, Molecular, Coenzymes, Computer Simulation, Culture Media, Dose-Response Relationship, Drug, Enzyme Activation, Geobacillus stearothermophilus, L-Lactate Dehydrogenase, Methanol, Models, Molecular, Mutagenesis, Site-Directed, NAD, NADP, Protein Engineering, Recombinant Proteins, Substrate Specificity
in
Protein Engineering
volume
12
issue
10
pages
6 pages
publisher
Oxford University Press
external identifiers
  • scopus:0032744506
ISSN
0269-2139
DOI
10.1093/protein/12.10.851
language
English
LU publication?
yes
id
814d2f0a-3e41-46c6-bd08-3975419c7248
date added to LUP
2016-04-18 15:57:57
date last changed
2017-07-02 04:50:21
@article{814d2f0a-3e41-46c6-bd08-3975419c7248,
  abstract     = {<p>L-lactate dehydrogenase (LDH) from Bacillus stearothermophilus is a redox enzyme which has a strong preference for NADH over NADPH as coenzyme. To exclude NADPH from the coenzyme-binding pocket, LDH contains a conserved aspartate residue at position 52. However, this residue is probably not solely responsible for the NADH specificity. In this report we examine the possibilities of altering the coenzyme specificity of LDH by introducing a range of different point mutations in the coenzyme-binding domain. Furthermore, after choosing the mutant with the highest selectivity for NADPH, we also investigated the possibility of further altering the coenzyme specificity by adding an organic solvent to the reaction mixture. The LDH mutant, I51K:D52S, exhibited a 56-fold increased specificity to NADPH over the wild-type LDH in a reaction mixture containing 15% methanol. Furthermore, the NADPH turnover number of this mutant was increased almost fourfold as compared with wild-type LDH. To explain the altered coenzyme specificity exhibited by the D52SI51K double mutant, molecular dynamics simulations were performed.</p>},
  author       = {Holmberg, Niklas and Ryde, U and Bülow, L},
  issn         = {0269-2139},
  keyword      = {Amino Acid Motifs,Binding Sites,Cloning, Molecular,Coenzymes,Computer Simulation,Culture Media,Dose-Response Relationship, Drug,Enzyme Activation,Geobacillus stearothermophilus,L-Lactate Dehydrogenase,Methanol,Models, Molecular,Mutagenesis, Site-Directed,NAD,NADP,Protein Engineering,Recombinant Proteins,Substrate Specificity},
  language     = {eng},
  number       = {10},
  pages        = {6--851},
  publisher    = {Oxford University Press},
  series       = {Protein Engineering},
  title        = {Redesign of the coenzyme specificity in L-lactate dehydrogenase from bacillus stearothermophilus using site-directed mutagenesis and media engineering},
  url          = {http://dx.doi.org/10.1093/protein/12.10.851},
  volume       = {12},
  year         = {1999},
}