Directed evolution of the substrate specificity of dialkylglycine decarboxylase

Jared L. Taylor, Joseph E. Price, Michael D. Toney

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~ 2.5-fold decrease in KM and ~ 2-fold increase in kcat, giving a modest ~ 5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

Original languageEnglish (US)
Pages (from-to)146-155
Number of pages10
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1854
Issue number2
DOIs
StatePublished - 2015

Fingerprint

2,2-dialkylglycine decarboxylase
Substrate Specificity
Mutation
Substrates
Enzymes
Decarboxylation
Catalytic Domain
Clone Cells
Denaturation
Pyridoxal Phosphate
Proton transfer
Kinetic parameters
Conformations
Molecular dynamics
Catalyst activity
Molecular Dynamics Simulation
Computer simulation
Protons

Keywords

  • Conformational change
  • Dialkylglycine decarboxylase
  • Directed evolution
  • Genetic selection
  • Pyridoxal phosphate
  • Stereoelectronic effect

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Analytical Chemistry
  • Molecular Biology

Cite this

Directed evolution of the substrate specificity of dialkylglycine decarboxylase. / Taylor, Jared L.; Price, Joseph E.; Toney, Michael D.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1854, No. 2, 2015, p. 146-155.

Research output: Contribution to journalArticle

Taylor, Jared L. ; Price, Joseph E. ; Toney, Michael D. / Directed evolution of the substrate specificity of dialkylglycine decarboxylase. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2015 ; Vol. 1854, No. 2. pp. 146-155.
@article{b7dc2e9377eb4b8d80a50934f0c45cbc,
title = "Directed evolution of the substrate specificity of dialkylglycine decarboxylase",
abstract = "Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~ 2.5-fold decrease in KM and ~ 2-fold increase in kcat, giving a modest ~ 5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.",
keywords = "Conformational change, Dialkylglycine decarboxylase, Directed evolution, Genetic selection, Pyridoxal phosphate, Stereoelectronic effect",
author = "Taylor, {Jared L.} and Price, {Joseph E.} and Toney, {Michael D.}",
year = "2015",
doi = "10.1016/j.bbapap.2014.12.003",
language = "English (US)",
volume = "1854",
pages = "146--155",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "2",

}

TY - JOUR

T1 - Directed evolution of the substrate specificity of dialkylglycine decarboxylase

AU - Taylor, Jared L.

AU - Price, Joseph E.

AU - Toney, Michael D.

PY - 2015

Y1 - 2015

N2 - Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~ 2.5-fold decrease in KM and ~ 2-fold increase in kcat, giving a modest ~ 5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

AB - Dialkylglycine decarboxylase (DGD) is an unusual pyridoxal phosphate dependent enzyme that catalyzes decarboxylation in the first and transamination in the second half-reaction of its ping-pong catalytic cycle. Directed evolution was employed to alter the substrate specificity of DGD from 2-aminoisobutyrate (AIB) to 1-aminocyclohexane-1-carboxylate (AC6C). Four rounds of directed evolution led to the identification of several mutants, with clones in the final rounds containing five persistent mutations. The best clones show ~ 2.5-fold decrease in KM and ~ 2-fold increase in kcat, giving a modest ~ 5-fold increase in catalytic efficiency for AC6C. Additional rounds of directed evolution did not improve catalytic activity toward AC6C. Only one (S306F) of the five persistent mutations is close to the active site. S306F was observed in all 33 clones except one, and the mutation is shown to stabilize the enzyme toward denaturation. The other four persistent mutations are near the surface of the enzyme. The S306F mutation and the distal mutations all have significant effects on the kinetic parameters for AIB and AC6C. Molecular dynamics simulations suggest that the mutations alter the conformational landscape of the enzyme, favoring a more open active site conformation that facilitates the reactivity of the larger substrate. We speculate that the small increases in kcat/KM for AC6C are due to two constraints. The first is the mechanistic requirement for catalyzing oxidative decarboxylation via a concerted decarboxylation/proton transfer transition state. The second is that DGD must catalyze transamination at the same active site in the second half-reaction of the ping-pong catalytic cycle.

KW - Conformational change

KW - Dialkylglycine decarboxylase

KW - Directed evolution

KW - Genetic selection

KW - Pyridoxal phosphate

KW - Stereoelectronic effect

UR - http://www.scopus.com/inward/record.url?scp=84919725189&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84919725189&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2014.12.003

DO - 10.1016/j.bbapap.2014.12.003

M3 - Article

C2 - 25500286

AN - SCOPUS:84919725189

VL - 1854

SP - 146

EP - 155

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 2

ER -