Attempts to develop an enzyme converting DHIV to KIV

Oki, Kenji; Lee, Frederick S.; Mayo, Stephen L.; Daggett, ed., Valerie

doi:10.1093/protein/gzz042

Title: Attempts to develop an enzyme converting DHIV to KIV

Abstract

Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants withmore » the desired activity were obtained.« less

Authors:

Oki, Kenji ^[1]; Lee, Frederick S. ^[2]; Mayo, Stephen L. ^[3]; Daggett, ed., Valerie

Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd., MC 114-96, Pasadena, CA 91125, USA, Science & Innovation Center, Mitsubishi Chemical Corporation, Yokohama 227-8502, Japan
Protabit LLC, 1010 Union St., Suite 110, Pasadena, CA 91101, USA
Division of Biology and Biological Engineering, California Institute of Technology, 1200 E. California Blvd., MC 114-96, Pasadena, CA 91125, USA, Division of Chemistry and Chemical Engineering, California Institute of Technology, Pasadena, CA 91125, USA

Publication Date:: 2019-12-23

Sponsoring Org.:: USDOE

OSTI Identifier:: 1580283

Grant/Contract Number:: SC0011396

Resource Type:: Published Article

Journal Name:: Protein Engineering, Design and Selection

Additional Journal Information:: Journal Name: Protein Engineering, Design and Selection Journal Volume: 32 Journal Issue: 6; Journal ID: ISSN 1741-0126

Publisher:: Oxford University Press

Country of Publication:: United Kingdom

Language:: English

Citation Formats


                    Oki, Kenji, Lee, Frederick S., Mayo, Stephen L., and Daggett, ed., Valerie. Attempts to develop an enzyme converting DHIV to KIV.  United Kingdom: N. p., 2019. 
Web.  doi:10.1093/protein/gzz042.

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                    Oki, Kenji, Lee, Frederick S., Mayo, Stephen L., & Daggett, ed., Valerie. Attempts to develop an enzyme converting DHIV to KIV.  United Kingdom.  https://doi.org/10.1093/protein/gzz042

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                    Oki, Kenji, Lee, Frederick S., Mayo, Stephen L., and Daggett, ed., Valerie. Mon .  
"Attempts to develop an enzyme converting DHIV to KIV".  United Kingdom.  https://doi.org/10.1093/protein/gzz042.

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@article{osti_1580283,

  title        = {Attempts to develop an enzyme converting DHIV to KIV},

  author       = {Oki, Kenji and Lee, Frederick S. and Mayo, Stephen L. and Daggett, ed., Valerie},

  abstractNote = {Abstract Dihydroxy-acid dehydratase (DHAD) catalyzes the dehydration of R-2,3-dihydroxyisovalerate (DHIV) to 2-ketoisovalerate (KIV) using an Fe-S cluster as a cofactor, which is sensitive to oxidation and expensive to synthesize. In contrast, sugar acid dehydratases catalyze the same chemical reactions using a magnesium ion. Here, we attempted to substitute the high-cost DHAD with a cost-efficient engineered sugar acid dehydratase using computational protein design (CPD). First, we tried without success to modify the binding pocket of a sugar acid dehydratase to accommodate the smaller, more hydrophobic DHIV. Then, we used a chemically activated substrate analog to react with sugar acid dehydratases or other enolase superfamily enzymes. Mandelate racemase from Pseudomonas putida (PpManR) and the putative sugar acid dehydratase from Salmonella typhimurium (StPutD) showed beta-elimination activity towards chlorolactate (CLD). CPD combined with medium-throughput selection improved the PpManR kcat/KM for CLD by four-fold. However, these enzyme variants did not show dehydration activity towards DHIV. Lastly, assuming phosphorylation could also be a good activation mechanism, we found that mevalonate-3-kinase (M3K) from Picrophilus torridus (PtM3K) exhibited adenosine triphosphate (ATP) hydrolysis activity when mixed with DHIV, indicating phosphorylation activity towards DHIV. Engineering PpManR or StPutD to accept 3-phospho-DHIV as a substrate was performed, but no variants with the desired activity were obtained.},

  doi          = {10.1093/protein/gzz042},

  journal      = {Protein Engineering, Design and Selection},

  number       = 6,

  volume       = 32,

  place        = {United Kingdom},

  year         = {2019},

  month        = {12}

}

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Title: Attempts to develop an enzyme converting DHIV to KIV

Abstract

Citation Formats

Site-directed mutagenesis by overlap extension using the polymerase chain reaction journal, April 1989

Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins journal, June 2011

Discovery of a Novel l -Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1 journal, May 2014

Identification and characterization of the bacterial d-gluconate dehydratase in Achromobacter xylosoxidans journal, August 2008

Evolution of Enzymatic Activities in the Enolase Superfamily: d -Mannonate Dehydratase from Novosphingobium aromaticivoran s † , ‡ journal, November 2007

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway journal, December 2013

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease journal, June 2012

Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway journal, March 2005

Computational redesign of endonuclease DNA binding and cleavage specificity journal, June 2006

Phosphate Recognition in Structural Biology journal, January 2007

Evolution of Enzymatic Activities in the Enolase Superfamily: l -Rhamnonate Dehydratase † ‡ journal, September 2008

Dramatic performance enhancements for the FASTER optimization algorithm journal, January 2006

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii journal, July 2009

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea journal, August 2012

Computational protein design promises to revolutionize protein engineering journal, January 2007

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Evolution of Enzymatic Activities in the Enolase Superfamily: l -Talarate/Galactarate Dehydratase from Salmonella typhimurium LT2 † , ‡ journal, August 2007

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SwiftLib: rapid degenerate-codon-library optimization through dynamic programming journal, December 2014

Enzyme-like proteins by computational design journal, November 2001

A method for extraction and determination of 2,4-dinitrophenylhydrazones of keto acids journal, May 1962

Evolution of Enzymatic Activities in the Enolase Superfamily: Identification of a “New” General Acid Catalyst in the Active Site of d -Galactonate Dehydratase from Escherichia coli journal, May 1999

Evolution of Enzymatic Activities in the Enolase Superfamily: Galactarate Dehydratase III from Agrobacterium tumefaciens C58 journal, June 2014

Discovery of Function in the Enolase Superfamily: d -Mannonate and d -Gluconate Dehydratases in the d -Mannonate Dehydratase Subgroup journal, April 2014

Establishment of Oxidative D-Xylose Metabolism in Pseudomonas putida S12 journal, March 2009

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles journal, November 2010

de novo computational enzyme design journal, October 2014

Evolution of Enzymatic Activities in the Enolase Superfamily: Characterization of the ( D )-Glucarate/Galactarate Catabolic Pathway in Escherichia coli † journal, October 1998

The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model journal, June 2005

Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity journal, January 2005

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe journal, July 2012

Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers journal, June 2015

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The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis journal, April 2016

Alteration of enzyme specificity by computational loop remodeling and design journal, May 2009

Backbone-dependent Rotamer Library for Proteins Application to Side-chain Prediction journal, March 1993

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis , journal, December 2009

UCSF Chimera?A visualization system for exploratory research and analysis journal, January 2004

Backrub-Like Backbone Simulation Recapitulates Natural Protein Conformational Variability and Improves Mutant Side-Chain Prediction journal, July 2008

Mechanism of Mevalonate Pyrophosphate Decarboxylase: Evidence for a Carbocationic Transition State journal, November 1994

A Carboxylate Oxygen of the Substrate Bridges the Magnesium Ions at the Active Site of Enolase: Structure of the Yeast Enzyme Complexed with the Equilibrium Mixture of 2-Phosphoglycerate and Phosphoenolpyruvate at 1.8 Å Resolution † , ‡ journal, January 1996

Site-directed mutagenesis by overlap extension using the polymerase chain reaction
journal, April 1989

Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins
journal, June 2011

Discovery of a Novel l -Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1
journal, May 2014

Identification and characterization of the bacterial d-gluconate dehydratase in Achromobacter xylosoxidans
journal, August 2008

Evolution of Enzymatic Activities in the Enolase Superfamily: d -Mannonate Dehydratase from Novosphingobium aromaticivoran s ^† ^, ^‡
journal, November 2007

Identification in Haloferax volcanii of Phosphomevalonate Decarboxylase and Isopentenyl Phosphate Kinase as Catalysts of the Terminal Enzyme Reactions in an Archaeal Alternate Mevalonate Pathway
journal, December 2013

Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease
journal, June 2012

Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway
journal, March 2005

Computational redesign of endonuclease DNA binding and cleavage specificity
journal, June 2006

Phosphate Recognition in Structural Biology
journal, January 2007

Evolution of Enzymatic Activities in the Enolase Superfamily: l -Rhamnonate Dehydratase ^† ^‡
journal, September 2008

Dramatic performance enhancements for the FASTER optimization algorithm
journal, January 2006

d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii
journal, July 2009

Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea
journal, August 2012

Computational protein design promises to revolutionize protein engineering
journal, January 2007

Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs
journal, April 2010

New algorithms and an in silico benchmark for computational enzyme design
journal, December 2006

Protein production by auto-induction in high-density shaking cultures
journal, May 2005

Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases: Structural Analysis of Mevalonate-3-Kinase
journal, December 2014

Evolution of Enzymatic Activities in the Enolase Superfamily: l -Talarate/Galactarate Dehydratase from Salmonella typhimurium LT2 ^† ^, ^‡
journal, August 2007

Formation of Isobutene from 3-Hydroxy-3-Methylbutyrate by Diphosphomevalonate Decarboxylase
journal, October 2010

SwiftLib: rapid degenerate-codon-library optimization through dynamic programming
journal, December 2014

Enzyme-like proteins by computational design
journal, November 2001

A method for extraction and determination of 2,4-dinitrophenylhydrazones of keto acids
journal, May 1962

Evolution of Enzymatic Activities in the Enolase Superfamily: Identification of a “New” General Acid Catalyst in the Active Site of d -Galactonate Dehydratase from Escherichia coli
journal, May 1999

Evolution of Enzymatic Activities in the Enolase Superfamily: Galactarate Dehydratase III from Agrobacterium tumefaciens C58
journal, June 2014

Discovery of Function in the Enolase Superfamily: d -Mannonate and d -Gluconate Dehydratases in the d -Mannonate Dehydratase Subgroup
journal, April 2014

Establishment of Oxidative D-Xylose Metabolism in Pseudomonas putida S12
journal, March 2009

Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles
journal, November 2010

de novo computational enzyme design
journal, October 2014

Evolution of Enzymatic Activities in the Enolase Superfamily: Characterization of the ( D )-Glucarate/Galactarate Catabolic Pathway in Escherichia coli ^†
journal, October 1998

The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model
journal, June 2005

Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity
journal, January 2005

HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe
journal, July 2012

Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers
journal, June 2015

Hydrophobic Nature of the Active Site of Mandelate Racemase ^†
journal, March 2004

The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis
journal, April 2016

Alteration of enzyme specificity by computational loop remodeling and design
journal, May 2009

Backbone-dependent Rotamer Library for Proteins Application to Side-chain Prediction
journal, March 1993

Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis ,
journal, December 2009

UCSF Chimera?A visualization system for exploratory research and analysis
journal, January 2004

Backrub-Like Backbone Simulation Recapitulates Natural Protein Conformational Variability and Improves Mutant Side-Chain Prediction
journal, July 2008

Mechanism of Mevalonate Pyrophosphate Decarboxylase: Evidence for a Carbocationic Transition State
journal, November 1994

A Carboxylate Oxygen of the Substrate Bridges the Magnesium Ions at the Active Site of Enolase: Structure of the Yeast Enzyme Complexed with the Equilibrium Mixture of 2-Phosphoglycerate and Phosphoenolpyruvate at 1.8 Å Resolution ^† ^, ^‡
journal, January 1996