Page 18 - ILLS Annual Report 2018

BIOLOGY AND HEALTH
Timothy C. Mueser. American Department of Chemistry and Biochemistry, University of Toledo, Toledo, Ohio USA
‘We use biophysical methods to analyse enzyme mechanisms and transient complexes. A recent emphasis on neutron crystallography has led us to use the microgravity environment
on the International Space Station to obtain large uniform crystals necessary for neutron diffraction.’
Re-writing biochemistry textbooks: neutrons observe protons critical for a vitamin B6-dependent enzyme function
Quasi-Laue diffractometer LADI-III
IMAGINE diffractometer at Oak Ridge National Laboratory
AUTHORS
S. Dajnowicz and T.C. Mueser (University of Toledo, USA)
R.C. Johnston, J.M. Parks, K.L. Weiss and A. Kovalevsky (Oak Ridge National Laboratory, Tennessee, USA)
M.P. Blakeley (ILL)
D.A. Keen (ISIS, UK)
O. Gerlits (University of Tennessee, Tennessee, USA)
ARTICLE FROM
Nat. Commun. (2017)—doi: 10.1038/s41467-017-01060
REFERENCES
[1] S. Dajnowicz, R.C. Johnston, J.M. Parks, M.P. Blakeley, D.A. Keen, K.L. Weiss, O. Gerlits, A. Kovalevsky and T.C. Mueser, Nat. Commun. 8 (2017) 955
[2] S. Dajnowicz, J.M. Parks, X. Hu, R.C. Johnston, A.Y. Kovalevsky and T.C. Mueser, ACS Catal. 8 (2018) 6733
[3] Lehninger Principles of Biochemistry, Eds. D.L. Nelson and M.M. Cox, by W. H. Freeman & Co. New York, 7th Edition, 2017, pp. 679−682.
Aspartate aminotransferase is crucial for amino acid metabolism, reversibly converting L-aspartic and L-glutamic amino acids. The study aimed to locate and reveal movement of hydrogen atoms within the enzyme’s
active site in two states: before and after substrate binding, called internal aldimine and external aldimine, respectively. Internal aldimine is a Schiff base generated when the PLP co-factor reacts with the enzyme’s catalytic lysine residue. External aldimine is similar, but the Schiff base connection is with the nitrogen of a substrate
amino acid (figure 1). For this study, room-temperature neutron crystallographic experiments were carried out
on the LADI-III diffractometer at the ILL and the IMAGINE diffractometer at the High Flux Isotope reactor at Oak Ridge National Laboratory. Neutron crystallography is uniquely able to provide accurate locations of hydrogen atoms in a biological macromolecule. To trap the enzyme in the substrate-bound, external aldimine state and directly visualise hydrogen atoms, a pseudo-substrate, α-methylaspartic acid, was used.
The conventional catalytic mechanism for aspartate aminotransferase is depicted in figure 2. It shows protonation of the Schiff base, the phenolic oxygen and the pyridine nitrogen in both the internal and external aldimine states, while the approaching substrate amino acid is depicted protonated at the N-terminal amino group. How the substrate is deprotonated to obtain a reactive amine and where hydrogens move when the latter attacks the carbon of the internal aldimine Schiff base to generate the external aldimine, was not known. Now, the two neutron structures of aspartate aminotransferase provide
the answers, requiring biochemists to re-think this enzyme’s catalytic mechanism.
The neutron structure of aspartate aminotransferase in
the internal aldimine state showed the presence of a hydrogen atom attached to the pyridine nitrogen of the PLP co-factor, whereas neither the phenolic oxygen nor the Schiff base nitrogen was protonated (figure 3). When the pseudo-substrate was soaked into the crystal, the external aldimine formed in which the Schiff base nitrogen
Vitamin B , or pyridoxine, and its derivatives are 6
involved in neurotransmitter synthesis, amino acid
metabolism and a plethora of other physiological
pathways in all known living organisms. When vitamin
B is derivatised inside cells, a very powerful co-factor, 6
pyridoxal 5’-phosphate or PLP, is produced, which is used by many enzymes to catalyse various biochemical transformations (figure 1). In fact, enzymes that are dependent on PLP for their function are responsible
for ~4 % of all classified enzymatic activity in nature. The PLP-dependent enzymes catalyse over 150
different chemical reactions, some of which do not even have analogues in organic chemistry. Moreover, the considerable dependence of micro-organisms on the action of these enzymes makes them good targets for novel antimicrobial drugs. The diverse chemistry performed by these enzymes is not well understood. Additionally, to understand PLP-dependent enzymes’ specificity, i.e. how they facilitate so many disparate chemical reactions using a single co-factor, locations and movement of hydrogen atoms need to be
mapped experimentally along the reaction pathways. Our recent published study [1] demonstrated the
power of macromolecular neutron crystallography to accurately pinpoint hydrogen atoms and to overturn long-held views on the catalytic mechanism of a PLP-dependent enzyme aspartate aminotransferase—
a classic biochemistry textbook example.
ANNUAL REPORT 2018