Constructing Enzyme Cascade Reactions toward Bioactive Compounds
Associate Professor (Biochemistry and Molecular Biology)
Primary Research Area
Other Area(s) of Interest
(Research Description PDF)
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.
Biocatalysis of Docetaxel (more here)
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 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).
Paclitaxel Biocatalysis of a Paclitaxel Analog: 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.
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. Bruening, ACS 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.
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)
|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|