Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Experiment Videos

Guided evolution of enzymes with new substrate specificities

A S el Hawrani1, R B Sessions, K M Moreton

  • 1Molecular Recognition Centre, Department of Biochemistry, Bristol, UK.

Journal of Molecular Biology
|November 22, 1996
PubMed
Summary

Altering surface loop amino acids in Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH) created stable enzyme variants with new substrate specificities. This protein engineering approach successfully generated novel enzymes for malate and phenylpyruvate metabolism.

Related Concept Videos

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by
Same author

Identification of key binding site residues of MCT1 for AR-C155858 reveals the molecular basis of its isoform selectivity.

The Biochemical journal·2015
Same author

Protein engineering applications of industrially exploitable enzymes: Geobacillus stearothermophilus LDH and Candida methylica FDH.

Biochemical Society transactions·2007
Same author

Amino acid pairing preferences in parallel beta-sheets in proteins.

Journal of molecular biology·2005
Same author

Crystal structure of Plasmodium berghei lactate dehydrogenase indicates the unique structural differences of these enzymes are shared across the Plasmodium genus.

Molecular and biochemical parasitology·2003
Same author

Protochlorophyllide oxidoreductase: a homology model examined by site-directed mutagenesis.

Proteins·2001
Same author

The A245K mutation exposes another stage of the bacterial L-lactate dehydrogenase reaction mechanism.

Biochemistry·2001

Area of Science:

  • Biochemistry
  • Protein Engineering
  • Enzyme Kinetics

Background:

  • Bacillus stearothermophilus L-lactate dehydrogenase (bsLDH) is a key enzyme in metabolic pathways.
  • Surface-exposed loops are often considered less critical for protein folding and stability.
  • Exploring sequence-function relationships in enzyme active sites is crucial for protein engineering.

Purpose of the Study:

  • To investigate the role of amino acids at positions 101 and 102 in the alpha E-beta D surface loop of bsLDH.
  • To engineer bsLDH variants with altered substrate specificities and enhanced catalytic properties.
  • To assess the impact of loop variation on enzyme stability and hydrodynamic properties.

Main Methods:

  • Construction of a gene library encoding all possible variants of amino acids at positions 101 and 102.

Related Experiment Videos

  • Expression and purification of bsLDH variants.
  • Thermal stability and hydrodynamic property assessments.
  • Enzyme activity assays for various substrates including malate and phenylpyruvate.
  • Molecular modeling to analyze active site interactions.

    • Main Results:

    • Most of the 38 examined bsLDH variants exhibited thermal stability and native-like hydrodynamic properties.
    • Enzyme variants demonstrated new substrate specificities, including for malate, phenyllactate, hydroxyisocaproate, and 4-phenyl-2-hydroxy-butanoate.
    • Specific amino acid substitutions at positions 101 and 102 correlated with altered substrate recognition, as predicted by molecular modeling.
    • A variant with Arg101Arg102 showed a 25% improvement in activity over the best previously reported synthetic malate dehydrogenase.
    • Unexpected substrate specificities were observed for certain variants, highlighting the complexity of enzyme-substrate interactions.

    Conclusions:

    • The alpha E-beta D surface loop sequence in bsLDH is not critical for protein folding or stability and can be extensively modified.
    • Protein engineering by varying surface loop residues is a viable strategy for creating enzymes with novel substrate specificities.
    • The study provides insights into the structural determinants of substrate recognition in NAD-dependent dehydrogenases.
    • Engineered bsLDH variants offer potential applications in biocatalysis and metabolic engineering.