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

Journal Article · Mon Dec 23 00:00:00 EST 2019 · Protein Engineering, Design and Selection

DOI:https://doi.org/10.1093/protein/gzz042· OSTI ID:1580283

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

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.

View Journal Article

Cite

Export

Save

Sponsoring Organization:: USDOE

Grant/Contract Number:: SC0011396

OSTI ID:: 1580283

Journal Information:: Protein Engineering, Design and Selection, Journal Name: Protein Engineering, Design and Selection Vol. 32 Journal Issue: 6; ISSN 1741-0126

Publisher:: Oxford University PressCopyright Statement

Country of Publication:: United Kingdom

Language:: English

Citation Metrics:

Cited by: 2 works

Citation information provided by
Web of Science

References (44)

Site-directed mutagenesis by overlap extension using the polymerase chain reaction Ho, Steffan N.; Hunt, Henry D.; Horton, Robert M. Gene, Vol. 77, Issue 1, p. 51-59 https://doi.org/10.1016/0378-1119(89)90358-2	journal	April 1989
Computational protein design: engineering molecular diversity, nonnatural enzymes, nonbiological cofactor complexes, and membrane proteins Saven, Jeffery G. Current Opinion in Chemical Biology, Vol. 15, Issue 3 https://doi.org/10.1016/j.cbpa.2011.03.014	journal	June 2011
Discovery of a Novel l -Lyxonate Degradation Pathway in Pseudomonas aeruginosa PAO1 Ghasempur, Salehe; Eswaramoorthy, Subramaniam; Hillerich, Brandan S. Biochemistry, Vol. 53, Issue 20 https://doi.org/10.1021/bi5004298	journal	May 2014
Identification and characterization of the bacterial d-gluconate dehydratase in Achromobacter xylosoxidans Kim, Seonghun; Lee, Sun Bok Biotechnology and Bioprocess Engineering, Vol. 13, Issue 4 https://doi.org/10.1007/s12257-008-0152-y	journal	August 2008
Evolution of Enzymatic Activities in the Enolase Superfamily: d -Mannonate Dehydratase from Novosphingobium aromaticivoran s ^† ^, ^‡ Rakus, John F.; Fedorov, Alexander A.; Fedorov, Elena V. Biochemistry, Vol. 46, Issue 45 https://doi.org/10.1021/bi701703w	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 VanNice, J. C.; Skaff, D. A.; Keightley, A. Journal of Bacteriology, Vol. 196, Issue 5 https://doi.org/10.1128/JB.01230-13	journal	December 2013
Structural, kinetic, and thermodynamic studies of specificity designed HIV-1 protease Alvizo, Oscar; Mittal, Seema; Mayo, Stephen L. Protein Science, Vol. 21, Issue 7 https://doi.org/10.1002/pro.2086	journal	June 2012
Identification and characterization of Sulfolobus solfataricusD-gluconate dehydratase: a key enzyme in the non-phosphorylated Entner–Doudoroff pathway Kim, Seonghun; Lee, Sun Bok Biochemical Journal, Vol. 387, Issue 1 https://doi.org/10.1042/BJ20041053	journal	March 2005
Computational redesign of endonuclease DNA binding and cleavage specificity Ashworth, Justin; Havranek, James J.; Duarte, Carlos M. Nature, Vol. 441, Issue 7093 https://doi.org/10.1038/nature04818	journal	June 2006
Phosphate Recognition in Structural Biology Hirsch, Anna K. H.; Fischer, Felix R.; Diederich, François Angewandte Chemie International Edition, Vol. 46, Issue 3 https://doi.org/10.1002/anie.200603420	journal	January 2007
Evolution of Enzymatic Activities in the Enolase Superfamily: l -Rhamnonate Dehydratase ^† ^‡ Rakus, John F.; Fedorov, Alexander A.; Fedorov, Elena V. Biochemistry, Vol. 47, Issue 38 https://doi.org/10.1021/bi800914r	journal	September 2008
Dramatic performance enhancements for the FASTER optimization algorithm Allen, Benjamin D.; Mayo, Stephen L. Journal of Computational Chemistry, Vol. 27, Issue 10 https://doi.org/10.1002/jcc.20420	journal	January 2006
d-Xylose Degradation Pathway in the Halophilic Archaeon Haloferax volcanii Johnsen, Ulrike; Dambeck, Michael; Zaiss, Henning Journal of Biological Chemistry, Vol. 284, Issue 40 https://doi.org/10.1074/jbc.M109.003814	journal	July 2009
Biochemical Characterization of Pantoate Kinase, a Novel Enzyme Necessary for Coenzyme A Biosynthesis in the Archaea Tomita, H.; Yokooji, Y.; Ishibashi, T. Journal of Bacteriology, Vol. 194, Issue 19 https://doi.org/10.1128/JB.06624-11	journal	August 2012
Computational protein design promises to revolutionize protein engineering Alvizo, Oscar; Allen, Benjamin D.; Mayo, Stephen L. BioTechniques, Vol. 42, Issue 1 https://doi.org/10.2144/000112336	journal	January 2007
Computational reprogramming of homing endonuclease specificity at multiple adjacent base pairs Ashworth, Justin; Taylor, Gregory K.; Havranek, James J. Nucleic Acids Research, Vol. 38, Issue 16 https://doi.org/10.1093/nar/gkq283	journal	April 2010
New algorithms and an in silico benchmark for computational enzyme design Zanghellini, Alexandre; Jiang, Lin; Wollacott, Andrew M. Protein Science, Vol. 15, Issue 12 https://doi.org/10.1110/ps.062353106	journal	December 2006
Protein production by auto-induction in high-density shaking cultures Studier, F. William Protein Expression and Purification, Vol. 41, Issue 1, p. 207-234 https://doi.org/10.1016/j.pep.2005.01.016	journal	May 2005
Structural analysis of mevalonate-3-kinase provides insight into the mechanisms of isoprenoid pathway decarboxylases: Structural Analysis of Mevalonate-3-Kinase Vinokur, Jeffrey M.; Korman, Tyler P.; Sawaya, Michael R. Protein Science, Vol. 24, Issue 2 https://doi.org/10.1002/pro.2607	journal	December 2014
Evolution of Enzymatic Activities in the Enolase Superfamily: l -Talarate/Galactarate Dehydratase from Salmonella typhimurium LT2 ^† ^, ^‡ Yew, Wen Shan; Fedorov, Alexander A.; Fedorov, Elena V. Biochemistry, Vol. 46, Issue 33 https://doi.org/10.1021/bi7008882	journal	August 2007
Formation of Isobutene from 3-Hydroxy-3-Methylbutyrate by Diphosphomevalonate Decarboxylase Gogerty, D. S.; Bobik, T. A. Applied and Environmental Microbiology, Vol. 76, Issue 24 https://doi.org/10.1128/AEM.01917-10	journal	October 2010
SwiftLib: rapid degenerate-codon-library optimization through dynamic programming Jacobs, Timothy M.; Yumerefendi, Hayretin; Kuhlman, Brian Nucleic Acids Research, Vol. 43, Issue 5 https://doi.org/10.1093/nar/gku1323	journal	December 2014
Enzyme-like proteins by computational design Bolon, D. N.; Mayo, S. L. Proceedings of the National Academy of Sciences, Vol. 98, Issue 25 https://doi.org/10.1073/pnas.251555398	journal	November 2001
A method for extraction and determination of 2,4-dinitrophenylhydrazones of keto acids Kawano, Chizoko; Katsuki, Hirohiko; Yoshida, Tsuya Analytical Biochemistry, Vol. 3, Issue 5 https://doi.org/10.1016/0003-2697(62)90064-7	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 Wieczorek, Stacey J.; Kalivoda, Kathryn A.; Clifton, James G. Journal of the American Chemical Society, Vol. 121, Issue 18 https://doi.org/10.1021/ja990500w	journal	May 1999
Evolution of Enzymatic Activities in the Enolase Superfamily: Galactarate Dehydratase III from Agrobacterium tumefaciens C58 Groninger-Poe, Fiona P.; Bouvier, Jason T.; Vetting, Matthew W. Biochemistry, Vol. 53, Issue 25 https://doi.org/10.1021/bi5005377	journal	June 2014
Discovery of Function in the Enolase Superfamily: d -Mannonate and d -Gluconate Dehydratases in the d -Mannonate Dehydratase Subgroup Wichelecki, Daniel J.; Balthazor, Bryan M.; Chau, Anthony C. Biochemistry, Vol. 53, Issue 16 https://doi.org/10.1021/bi500264p	journal	April 2014
Establishment of Oxidative D-Xylose Metabolism in Pseudomonas putida S12 Meijnen, J. -P.; de Winde, J. H.; Ruijssenaars, H. J. Applied and Environmental Microbiology, Vol. 75, Issue 9 https://doi.org/10.1128/AEM.02713-08	journal	March 2009
Experimental library screening demonstrates the successful application of computational protein design to large structural ensembles Allen, B. D.; Nisthal, A.; Mayo, S. L. Proceedings of the National Academy of Sciences, Vol. 107, Issue 46 https://doi.org/10.1073/pnas.1012985107	journal	November 2010
de novo computational enzyme design Zanghellini, Alexandre Current Opinion in Biotechnology, Vol. 29 https://doi.org/10.1016/j.copbio.2014.03.002	journal	October 2014
Evolution of Enzymatic Activities in the Enolase Superfamily: Characterization of the ( D )-Glucarate/Galactarate Catabolic Pathway in Escherichia coli ^† Hubbard, Brian K.; Koch, Marjan; Palmer, David R. J. Biochemistry, Vol. 37, Issue 41 https://doi.org/10.1021/bi981124f	journal	October 1998
The Substrate Spectrum of Mandelate Racemase: Minimum Structural Requirements for Substrates and Substrate Model Felfer, Ulfried; Goriup, Marian; Koegl, Marion F. Advanced Synthesis & Catalysis, Vol. 347, Issue 7-8 https://doi.org/10.1002/adsc.200505012	journal	June 2005
Divergent evolution in the enolase superfamily: the interplay of mechanism and specificity Gerlt, John A.; Babbitt, Patricia C.; Rayment, Ivan Archives of Biochemistry and Biophysics, Vol. 433, Issue 1 https://doi.org/10.1016/j.abb.2004.07.034	journal	January 2005
HmgR, a key enzyme in the mevalonate pathway for isoprenoid biosynthesis, is essential for growth of Listeria monocytogenes EGDe Heuston, Sinead; Begley, Máire; Davey, Martin S. Microbiology, Vol. 158, Issue 7 https://doi.org/10.1099/mic.0.056069-0	journal	July 2012
Metabolic engineering of Saccharomyces cerevisiae for production of butanol isomers Generoso, Wesley Cardoso; Schadeweg, Virginia; Oreb, Mislav Current Opinion in Biotechnology, Vol. 33 https://doi.org/10.1016/j.copbio.2014.09.004	journal	June 2015
Hydrophobic Nature of the Active Site of Mandelate Racemase ^† St. Maurice, Martin; Bearne, Stephen L. Biochemistry, Vol. 43, Issue 9 https://doi.org/10.1021/bi036207x	journal	March 2004
The MPI bioinformatics Toolkit as an integrative platform for advanced protein sequence and structure analysis Alva, Vikram; Nam, Seung-Zin; Söding, Johannes Nucleic Acids Research, Vol. 44, Issue W1 https://doi.org/10.1093/nar/gkw348	journal	April 2016
Alteration of enzyme specificity by computational loop remodeling and design Murphy, P. M.; Bolduc, J. M.; Gallaher, J. L. Proceedings of the National Academy of Sciences, Vol. 106, Issue 23 https://doi.org/10.1073/pnas.0811070106	journal	May 2009
Backbone-dependent Rotamer Library for Proteins Application to Side-chain Prediction Dunbrack, Roland L.; Karplus, Martin Journal of Molecular Biology, Vol. 230, Issue 2 https://doi.org/10.1006/jmbi.1993.1170	journal	March 1993
Computation-Facilitated Assignment of the Function in the Enolase Superfamily: A Regiochemically Distinct Galactarate Dehydratase from Oceanobacillus iheyensis , Rakus, John F.; Kalyanaraman, Chakrapani; Fedorov, Alexander A. Biochemistry, Vol. 48, Issue 48 https://doi.org/10.1021/bi901731c	journal	December 2009
UCSF Chimera?A visualization system for exploratory research and analysis Pettersen, Eric F.; Goddard, Thomas D.; Huang, Conrad C. Journal of Computational Chemistry, Vol. 25, Issue 13 https://doi.org/10.1002/jcc.20084	journal	January 2004
Backrub-Like Backbone Simulation Recapitulates Natural Protein Conformational Variability and Improves Mutant Side-Chain Prediction Smith, Colin A.; Kortemme, Tanja Journal of Molecular Biology, Vol. 380, Issue 4 https://doi.org/10.1016/j.jmb.2008.05.023	journal	July 2008
Mechanism of Mevalonate Pyrophosphate Decarboxylase: Evidence for a Carbocationic Transition State Dhe-Paganon, Sirano; Magrath, Joe; Abeles, Robert H. Biochemistry, Vol. 33, Issue 45 https://doi.org/10.1021/bi00249a023	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 ^† ^, ^‡ Larsen, Todd M.; Wedekind, Joseph E.; Rayment, Ivan Biochemistry, Vol. 35, Issue 14 https://doi.org/10.1021/bi952859c	journal	January 1996

Similar Records

Evolution of Enzymatic Activities int he Enolase Superfamily: L-Talarate/Galactarate Dehydratase from Salmonella typhimurium LT2

Journal Article · Mon Jan 01 00:00:00 EST 2007 · Biochemistry · OSTI ID:1580283

Yew, W; Fedorov, A; Fedorov, E; +2 more

Evolution of Enzymatic Activities in the Enolase Superfamily: L-Fuconate Dehydratase from Xanthomonas campestris

Journal Article · Sun Jan 01 00:00:00 EST 2006 · Biochemistry · OSTI ID:1580283

Yew, W; Fedorov, A; Fedorov, E; +4 more

Evolution of Enzymatic Activities in the Enolase Superfamily: D-Tartrate Dehydratase from Bradyrhizobium japonicum

Journal Article · Sun Jan 01 00:00:00 EST 2006 · Biochemistry · OSTI ID:1580283

Yew, W; Fedorov, A; Fedorov, E; +3 more

Title: Attempts to develop an enzyme converting DHIV to KIV

Citation Formats

References (44)

Similar Records

Related Subjects