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Kevin Walker

Constructing Enzyme Cascade Reactions toward Bioactive Compounds

Kevin Walker Kevin Walker

Associate Professor (in Chemistry and in Biochemistry and Molecular Biology)

208 CEM

517-353-1112


Research webpage

Google Scholar Link


Research Area

Analytical (An)

Biological (Bi)

Chemical Education (CE)

Chemical Physics (CP)

Forensic (F)

Inorganic (In)

Material (Ma)

Nuclear (Nu)

Organic (Or)

Organometallic (Om)

Physical (Ph)

Theoretical and Computational (Th)

Area(s) of Interest

Analytical (An)

Biological (Bi)

Chemical Education (CE)

Chemical Physics (CP)

Forensic (F)

Geochemistry (G)

Inorganic (In)

Material (Ma)

Nuclear (Nu)

Organic (Or)

Organometallic (Om)

Physical (Ph)

Theoretical and Computational (Th)

Research

(Research Description PDF)

A More Complete Research Description (click here)


We use interdisciplinary methods to evaluate enzyme catalysts from various sources, such as bacteria, plants, and yeast, with non-natural substrates. Our vision is to transform natural compounds or synthetically-derived chemicals to novel products. Transfer of the genes encoding these enzymes into a chassis organism can potentially make various bioactive molecules in vivo or in vitro.




An MIO-dependent aminomutase from Taxus canadensis (TcPAM) was repurposed to transfer the amino group irreversibly from (2S)-styryl-α-alanine to exogenously supplied trans-3-phenylglycidate enantiomers. TcPAM converted (2S,3R)-3-phenylglycidate to (2S)-anti-phenylserine predominantly (89%) and (2R,3S)-3-phenylglycidate to (2R)-anti-phenylserine (88%) over their antipodes, with inversion of the configuration at Cα in each case.

Bifurcation path TcPAM with epoxides


Recycling CoA in the Biocatalysis of Surrogate Taxanes to Access New Generation Bioactive Compounds

 


Biocatalysis of Arylserines and Arylisoserines from Epoxides by an MIO α/β-Aminomutase and a Mild Amine-Group Donor (more here)

Epoxide Isomers Docked in TcPAM 

We diverged from a fixed program of using MIO-aminomutase/lyases to aminate cinnamic acids and describe a first account of employing an MIO-aminomutase (TcPAM) from Taxus plants to convert previously untested epoxy acid substrates to bifunctional hydroxy amino acids. Arylserines were made more abundantly over the arylisoserines when styrylalanine was used instead of ammonium salts, to selectively transaminate the MIO group of TcPAM. This mild amine group delivery to the oxirane avoided nonenzymatic ring-opening of the epoxides.


Taxane analogues (docetaxel, paclitaxel, cabazitaxel, paclitaxel C, and tesetaxel) are used 1) for breast, ovarian, and prostate cancers, 2) to stem complications from stent implants in heart surgery, and 3) to work potentially as neuroprotectants against stroke. Current methods to make docetaxel still use an 11 to 12-step semisynthesis, which involves protecting group chemistry that compromises yields and reduces atom economy.

We use regioselective biocatalysts (Taxus Acyltransferases (AT) and Bacterial CoA Ligases) to bypass protecting group chemistry to make the drug docetaxel.

Streamlined 3-Step Biocatalysis of Docetaxel: An alternative to make docetaxel:

Coupling acyltransferases with CoA ligases (above) provides a Green source of docetaxel and its drug analogues.


Paclitaxel (Taxol) Pathway Aminomutase. A Taxus phenylalanine aminomutase (TcPAM) converts (2S)-α-phenylalanine ((2S)-α-Phe) to (3R)-β-Phe and lies on the paclitaxel (Taxol™) biosynthetic pathway in Taxus plants.

To understand how to use TcPAM chemistry to biocatalyze β-amino acids, it is necessary to understand the subtleties of its mechanism

The aminomutase forms a transient MIO-NH2 adduct with a finite lifetime. The lifetime of adduct was unknown for TcPAM or any of the several enzymes in this family until we used stopped-flow monitoring of product release to measure the exponential burst phase at presteady state.


Andrimid Pathway Aminomutase. TcPAM converts (2S)-α-Phe to (3R)-β-Phe, while PaPAM on the andrimid biosynthetic pathway converts the same substrate to (3S)-β- Phe. We used the TcPAM structure in complex with (E)-cinnamate, which functions as both a substrate and an intermediate, and the PaPAM structure to account for the distinct β-amino acid stereochemistries. TcPAM must rotate/flip the cinnamate skeleton 180° before exchange and PaPAM must hold the intermediate stationary before rebinding of the NH2/H pair to the cinnamate.

 

Phe455 (spheres) in PaPAM shown displacing the phenylpropanoate ligand (green), preventing a bidentate linkage (magenta) with Arg323. This trajectory may explain the different product stereochemistries of the two enzymes—TcPAM forms a bidentate complex with its substrate.


In vivo Biocatalysis of β-Amino Acids

Our goal is to use this mechanistic information to repurpose these aminomutases to produce value-added phenylpropanoids.

PaPAM biocatalyzed various phenyl- (and heteryl-) β-amino acids from their corresponding α-amino acids.


A new graduate student can embark on studies involving organic chemistry synthesis of novel surrogate substrates. Other areas of training include molecular cloning techniques, expression of various enzymes in E. coli, and assay development. Included are basic biochemical applications and molecular engineering approaches related to enzyme kinetics, enzyme purification and characterization, and various analytical techniques (such as NMR, GC/ MS, LC-MS(/MS), and X-ray crystallography).

 

Selected Publications

For Complete Listing of Publications and Patents (click here)

PATENT: Developing a Semibiocatalytic Process toward Regioisomerically Enriched Alkyl Pyrazines, G. Attanayake; G. Mao; K.D. Walker. Under Patent Review, 2021.


Intermolecular amine transfer to enantioenriched trans-3-phenylglycidates by an α/β-aminomutase to access both anti-phenylserine isomers, P.K. Shee; H. Yan; K.D. Walker. ACS Catal. 2020, 10, 15071-15082DOI: 10.1021/acscatal.0c03977



CoA Recycling by a Benzoate Coenzyme A Ligase in Cascade Reactions with Aroyltransferases to Biocatalyze Paclitaxel Analogs, S.A. Sullivan, ; I.N. Nawarathne; K.D. Walker. Arch. Biochem. Biophys. 2020, Arch. Biochem. Biophys. 2020, 683, 108276. DOI: 10.1016/j.abb.2020.108276


Exploring the Scope of an α/β-Aminomutase for the Amination of Cinnamate Epoxides to Arylserines and Arylisoserines, P.K. Shee; N.D. Ratnayake; T. Walter; O. Goethe; E.N. Onyeozili; K.D. Walker. ACS Catal. 2019, 9, 7418-7430. DOI: 10.1021/acscatal.9b01557


Understanding Which Residues of the Active Site and Loop Structure of a Tyrosine Aminomutase Define its Mutase and Lyase Activities, G. Attanayake; T. Walter; K. D. Walker. Biochemistry (ACS), 2018, 57 (25), 3503–3514. DOI: 10.1021/acs.biochem.8b00269 in Special Issue: Current Topics in Mechanistic Enzymology 


Biocatalysis of a Paclitaxel Analogue: Conversion of Baccatin III to N-Debenzoyl-N-(2-furoyl)paclitaxel and Characterization of an Amino Phenylpropanoyl CoA Transferase. C.K. Thornburg; T. Walter;  K.D. Walker. Biochemistry (ACS) 2017, 56 (44), 5920–5930. DOI: 10.1021/acs.biochem.7b00912.


Paclitaxel Biosynthesis: Adenylation and Thiolation Domains of an NRPS TycA PheAT Module Produce Various Arylisoserine CoA Thioesters. R. Muchiri; K.D. Walker. 2017, Biochemistry (ACS)  56 (10), 1415–1425: DOI: 10.1021/acs.biochem.6b01188.



Identification and characterization of the missing phosphatase on the riboflavin biosynthesis pathway in Arabidopsis thaliana. N. Sa; R. Rawat; C.K. Thornburg; K.D. Walker; S. Roje. 2016, Plant J. 88 (5), 705–716 DOI: 10.1111/tpj.13291 Featured Article. Cover Art

Mutation of aryl binding-pocket residues results in an unexpected activity switch in an Oryza sativa tyrosine aminomutase. T. Walter; D. Wijewardena; K.D. Walker, Biochemistry, 2016, 55 (25), 3497–3503 DOI: 10.1021/acs.biochem.6b00331.


Layer-by-layer deposition with polymers containing nitrilotriacetate, a convenient route to fabricate metal- and protein-binding films​. S. Wijeratne; W. Liu; J. Dong; W. Ning; N.D. Ratnayake; K.D. Walker; M.L. BrueningACS Appl. Mater. Interfaces, 2016, 8, 10164–10173 DOI: 10.1021/acsami.6b00896


A Tyrosine Aminomutase from Rice (Oryza sativa) Isomerizes (S)-α- to (R)-β-Tyrosine with Unique High Enantioselectivity and Retention of Configuration. T. Walter; Z. King; K. D. Walker, Biochemistry, 2016, 55 (1), 1–4 DOI: 10.1021/acs.biochem.5b01331


Whole-cell biocatalytic production of variously substituted β-aryl- and β-heteroaryl-β-amino acids, N. D. Ratnayake; C. Theisen.; T. Walter; K. D. Walker, J. Biotechnol., 2016, 217, 12–21.  DOI: 10.1016/j.jbiotec.2015.10.012


Kinetically and crystallographically guided mutations of a benzoate CoA ligase (BadA) elucidate mechanism and expand substrate permissivity, C. K. Thornburg; S. Wortas-Strom; M. Nosrati; J. H. Geiger; K. D. Walker, Biochemistry, 2015, 54(40), 6230–6242. DOI: 10.1021/acs.biochem.5b00899


Ring‑substituted α‑arylalanines for probing substituent effects on the isomerization reaction catalyzed by an aminomutase, N.D. Ratnayake; N. Liu; L. A. Kuhn; K. D. Walker, ACS Catalysis, 2014, 4(9), 3077.


Genome sequencing and analysis of the paclitaxel-producing endophytic fungus Penicillium aurantiogriseum, Y. Yang; H. Zhao; R. A. Barrero; B. Zhang; G. Sun; I. W. Wilson; F. Xie; K. D. Walker; J. W. Parks; R. Bruce; et al., BMC Genomics, 2014, 15(1):69.


A bacterial tyrosine aminomutase proceeds through retention or inversion of stereochemistry to catalyze its isomerization reaction, U. Wanninayake; K.D. Walker, J. Am. Chem.Soc., 2013, 135(30), 11193.


Assessing the deamination rate of a covalent aminomutase adduct by burst phase analysis, U. Wanninayake; K.D. Walker, Biochemistry, 2012, 51(26), 5226.


Taxol biosynthesis: Tyrocidine synthetase A catalyzes the production of phenylisoserinyl CoA and other amino phenylpropanoyl thioesters, R. Muchiri; K.D. Walker, Chem. Biol., 2012, 19(6), 679.


Insights into the mechanistic pathway of the Pantoea agglomerans phenylalanine aminomutase, S. Strom; U. Wanninayake; N. D. Ratnayake; K. D. Walker; J. H. Geiger, Ang. Chem. Int. Ed., 2012, 51(12), 2898.


(S)-Styryl-α-alanine used to probe the intermolecular mechanism of an intramolecular MIO-aminomutase, U. Wanninayake; Y. Deporre; M. Ondari; K. D. Walker, Biochemistry, 2011, 50(46), 10082.

CV

 

Chemistry, Biochemistry, Molecular Biology, and Biocatalysis

B.S., 1988, Univ. of Washington

Research Chemist, 1988-1990, FDA (Bothell, WA)

Ph.D., 1997, Univ. of Washington

NIH Postdoc. Research Fellow, 1997-2000, Institute of Biological Chemistry, Washington State Univ.

Research Assistant Professor, 2000-2003, Institute of Biological Chemistry, Washington State Univ.

Curriculum Vitae (click here)

Awards/Honors

2021 MTRAC AgBio Innovation Challenge Award MSU Innovation Center
2011 Outstanding Graduate Advisor
2006 Neish Young Investigator Award Phytochemical Society of North America
1997 Ph.D. University of Washington, Seattle
1988 Bachelor of Science University of Washington, Seattle