Research

My primary research interests lie in the broadly defined area of mechanistic enzymology. Two representative projects, which focus on distinctly different aspects of this field, are as follows:

Structure and function of the mandelate pathway enzymes.

This pathway, which permits a variety of pseudomonads to use the enantiomers of mandelic acid as sole carbon source, is thought to be a paradigm for the study of functional evolution. The known pathway comprises five enzymes, each of which utilizes quite different chemistry. We are using a combination X-ray crystallography, site-direct mutagenesis and mechanistic studies to understand the chemistry behind each of these enzymes. Armed with this knowledge we aim to use directed evolution to develop a new pathway, the lactate pathway. In the new pathway, instead of utilizing aromatic substrates, each enzyme will use the corresponding aliphatic substrate. In some instances the evolutionary process should be relatively simple, needing only minor changes in substrate specificity. In other cases the new enzyme may need to employ new chemistry.

Inhibition of the epinephrine synthesizing enzyme, phenylethanolamine N-methyltransferase (PNMT).

While the role of peripheral epinephrine (adrenaline) is reasonably well understood its role in the CNS is not so well defined. PNMT inhibitors could be used clarify its role, particularly in the central control of hypertension. Unfortunately it is hard to get a clear-cut picture of CNS epinephrine effects as most of the currently available inhibitors also interact with adrenoceptors. We are using a combination of QSAR methods, kinetic and mutagenic analysis as well as structure-based design to develop potent and specific PNMT inhibitors. Initially these will be used to delineate the role of CNS epinephrine but, ultimately, we hope they will be useful as antihypertensives with a novel mode of action.

Education

• B.Sc., (Honors), La Trobe University, Australia, 1978
• Ph.D., La Trobe University, Australia, 1984
• Post-Doctoral Fellow, University of California-Berkeley, 1983-1984

Publications & Professional Activity

R. F. Pratt and M. J. McLeish “The structural relationship between the active sites of β-lactam-recognizing and amidase signature enzymes: convergent evolution?” Biochemistry 2010, 49, 9688-9697.

N. Drinkwater, H. Vu, K. M. Lovell, K. R. Criscione, B. M. Collins, T. E. Prisinzano, S.-A. Poulsen, M. J. McLeish, G. L. Grunewald and J. L. Martin “Fragment-based screening by X-ray crystallography, MS and isothermal titration calorimetry to identify PNMT (phenylethanolamine N-methyltransferase) inhibitors” Biochemical Journal 2010, 431, 51-61.

G. S. Brandt, M. M. Kneen, G. A. Petsko, D. Ringe and M. J. McLeish “Active-site engineering of benzaldehyde lyase shows that a point mutation can confer both new reactivity and susceptibility to mechanism-based inhibition” Journal of the American Chemical Society 2010, 132, 438-439.

A. Yep and M. J. McLeish “Evolution of a benzoylformate decarboxylase variant with enhanced pyruvate decarboxylase activity” Biochemistry 2009, 48, 8387-8395.

N. Drinkwater, C. L. Gee, M. Puri, K. R. Criscione, M. J. McLeish, G. L. Grunewald and J. L. Martin “Molecular recognition of physiological substrate noradrenaline by the adrenaline synthesising enzyme PNMT and factors influencing its methyltransferase activity” Biochemical Journal 2009, 422, 463-471.

P. Georgieva, Q. Wu, M. J. McLeish and F. Himo “The reaction mechanism of phenylethanolamine N-Methyltransferase: A density functional theory study” Biochimica et Biophysica Acta - Proteins and Proteomics 2009, 1794, 1831-1837.

P. F. Wang, A. Yep, G. L. Kenyon and M. J. McLeish “Using directed evolution to probe the substrate specificity of mandelamide hydrolase” Protein Engineering, Design and Selection 2009, 22, 103-110.

G. S. Brandt, M. M. Kneen, S. Chakraborty, A. T. Baykal, N. Nemeria, A. Yep, D. I. Ruby, G. Petsko, G. L. Kenyon, M. J. McLeish, F. Jordan and D. Ringe ”Snapshot of a reaction intermediate: analysis of benzoylformate decarboxylase in complex with a benzoylphosphonate inhibitor” Biochemistry 2009, 48, 3247-3257.

S. Chakraborty, N. Nemeria, G. S. Brandt, M. M. Kneen, A. Yep, M. J. McLeish, G. L. Kenyon, G. Petsko, D. Ringe and F. Jordan “Detection and time-course of formation of all thiamin diphosphate-bound covalent intermediates derived from a chromophoric substrate analogue on benzoylformate decarboxylase” Biochemistry 2009, 48, 981-994.

Q. Wu, J. M. Caine, S. A. Thomson, M. Slavica, G. L. Grunewald and M. J. McLeish “Time-dependent inactivation of human phenylethanolamine N-methyltransferase by 7-isothiocyanatotetrahydroisoquinoline” Bioorganic and Medicinal Chemistry Letters 2009, 19, 1071-1074.

G. Brandt, N. Nemeria, S. Chakraborty, M. J. McLeish, A. Yep, G. L. Kenyon, G. Petsko, F. Jordan and D. Ringe "Probing the active center of benzaldehyde lyase with substitutions and the pseudo-substrate analogue benzoylphosphonic acid methyl ester" Biochemistry 2008, 47, 7734-7743.

S. Chakraborty, N. Nemeria, A. Yep, M. J. McLeish, G. L. Kenyon and F. Jordan "The mechanism of benzaldehyde lyase studied via thiamin diphosphate-bound Intermediates and kinetic isotope effects" Biochemistry 2008, 47, 3800-3809.

A. Yep, G. L. Kenyon and M. J. McLeish "Saturation mutagenesis of putative catalytic residues of benzoylformate decarboxylase provides a challenge to the accepted mechanism" Proceedings of the National Academy of Sciences USA 2008, 105, 5733- 5738.

C. K. Yeung, G. L. Kenyon and M. J. McLeish "Physical, kinetic and spectrophotometric studies of a NAD(P)+-dependent benzaldehyde dehydrogenase from Pseudomonas putida ATCC 12633" Biochimica et Biophysica Acta - Proteins and Proteomics 2008, 1784, 1248-1255.

N. Nemeria, L. Korotchkina, M. J. McLeish, G. L. Kenyon, M. S. Patel and F. Jordan "Elucidation of the chemistry of enzyme-bound thiamin diphosphate prior to substrate binding: defining internal equilibria among tautomeric and ionization states " Biochemistry 2007, 46, 10739-10744.

C. Saehuan, T. Rojanarata, S. Wiyakrutta, M. J. McLeish and V. Meevootisom "Isolation and characterization of a benzoylformate decarboxylase and a NAD+/NADP+-dependent benzaldehyde dehydrogenase involved in D-phenylglycine metabolism in Pseudomonas stutzeri ST-201" Biochimica et Biophysica Acta 2007, 1770, 1585-1592.

C. L. Gee, N. Drinkwater, J. D. A. Tyndall, G. L. Grunewald, Q. Qian Wu, M. J. McLeish and J. L. Martin "Enzyme adaptation to inhibitor binding: A cryptic binding site in phenylethanolamine N-methyltransferase" Journal of Medicinal Chemistry 2007, 50, 4845-4853.

A. K. Bera, L. S. Polovnikova, J. Roestamadji, T. S. Widlanski, G. L. Kenyon, M. J. McLeish and M. S. Hasson "Mechanism-based inactivation of benzoylformate decarboxylase, a thiamin diphosphate-dependent enzyme" Journal of the American Chemical Society 2007, 129, 4120-4121.

E. Janzen, M. Müller, D. Kolter-Jung, M. M. Kneen, M. J. McLeish and M. Pohl "Characterization of benzaldehyde lyase from Pseudomonas fluorescens - a versatile enzyme for asymmetric C-C-bond formation" Bioorganic Chemistry 2006, 34, 345- 361.

M. J. McLeish and G. L. Kenyon "Using site-directed mutagenesis to elucidate structure and mechanism in creatine kinase" In Creatine Kinase; Vial, C., Ed.; Nova Science, 2006, pp. 27-68.

A. Yep, G. L. Kenyon and M. J. McLeish "Determinants of substrate specificity in KdcA, a thiamin diphosphate-dependent decarboxylase" Bioorganic Chemistry 2006, 34, 325-336.

P.-F. Wang, A. J. Flynn, M. M. Naor, J. H. Jensen, G. Cui, K. M. Merz Jr., G. L. Kenyon and M. J. McLeish "Exploring the role of the active site cysteine in human muscle creatine kinase" Biochemistry 2006, 45, 11464-11472.

P.-F. Wang, G. L. Kenyon and M. J. McLeish "Heterogeneity of Escherichia coliexpressed human muscle creatine kinase" IUBMB Life 2006, 58, 421-428.

A. D. Andricopulo, M. B. Akoachere, R. Krogh, C. Nickel, M. J. McLeish, G. L. Kenyon, L. D. Arscott, C. H. J. Williams, E. Davioud-Charvet and K. Becker "Specific inhibitors of Plasmodium falciparum thioredoxin reductase as potential antimalarial agents" Bioorganic and Medicinal Chemistry Letters 2006, 16, 2283-2292.

C. L. Gee, J. D. A. Tyndall, G. L. Grunewald, Q. Wu, M. J. McLeish and J. L. Martin "Binding mode of methyl acceptor substrates to the adrenaline-synthesizing enzyme phenylethanolamine N-methyltransferase: Implications for catalysis" Biochemistry 2005, 44, 16875-16885.

M. M. Kneen, I. D. Pogozheva, G. L. Kenyon and M. J. McLeish "Exploring the active site of benzaldehyde lyase by modeling and mutagenesis" Biochimica et Biophysica Acta - Proteins and Proteomics 2005, 1753, 263-271.

Q. Wu, C. L. Gee, F. Lin, J. D. Tyndall, J. L. Martin, G. L. Grunewald and M. J. McLeish "Structural, mutagenic and kinetic analysis of the binding of substrates and inhibitors of human phenylethanolamine N-methyltransferase" Journal of Medicinal Chemistry 2005, 48, 7243-7252

P. Siegert, M. J. McLeish, M. Baumann, M. M. Kneen, G. L. Kenyon and M. Pohl "Exchanging the substrate specificities of pyruvate decarboxylase from Zymomonas mobilis and benzoylformate decarboxylase from Pseudomonas putida" Protein Engineering, Design and Selection 2005, 18, 345-357.

P.-F. Wang, A. D. Flynn, M. J. McLeish and G. L. Kenyon "Loop movement and catalysis in creatine kinase" IUBMB Life 2005, 57, 355-362.

M. J. McLeish and G. L. Kenyon "Relating structure to mechanism in creatine kinase" Critical Reviews in Biochemistry and Molecular Biology 2005, 40, 1-20.

C. L. Gee, A. Nourse, A.-Y. Hsin, Q. Wu, J. D. Tyndall, G. L. Grunewald, M. J. McLeish and J. L. Martin "Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active" Biochimica et Biophysica Acta - Proteins and Proteomics 2005, 1750, 82-92.