My group is working to develop NMR-assisted crystallography – the synergistic combination of
solid-state nuclear magnetic resonance, X-ray crystallography, and computational chemistry – as an
atomic-resolution probe of enzyme active sites, capable of defining the position of all atoms, including
hydrogens. By locating hydrogen atoms, this technique provides the often critical missing chemical
information necessary to link structure and mechanism, as well as providing crucial information for the rational
design of therapeutics. The approach is three-fold: X-ray crystallography is used to provide a coarse structural
framework upon which chemically-detailed models of the active site are built using computational chemistry,
and various active site chemistries explored; these models can be quantitatively distinguished by comparing
their predicted NMR chemical shifts with the results from solid-state NMR experiments. Provided a sufficient
number of chemical shift restraints are measured within the active site, NMR-assisted crystallography can
uniquely identify the structure. The targeted systems include pyridoxal-5’-phosphate (PLP)-dependent
enzymes, which have been implicated in numerous health conditions and as targets for treating diseases, and
the ß-Lactamases, which mediate antibiotic resistance to ß-lactam antibiotics.
The family of PLP-dependent enzymes are involved in the metabolism of amino acids and other amine-
containing biomolecules. This single cofactor can participate in a diverse array of chemical transformations,
including racemization, transamination, a/ß-decarboxylation, and a/ß/¿- elimination and substitution.
Understanding how active sites fine-tune the same cofactor for such varied reactions is a primary objective of
this proposal. To accomplish this understanding, NMR-assisted crystallography is employed to characterize
these enzymatic transformations with atomic resolution. In tryptophan synthase, this allows us to peer along
the reaction coordinates into and out of multiple intermediates. Here the protonation states complete the
chemical picture for why, for example, specific inhibitors such as benzimidazole are unable to react to form a
covalent bond as it is held in the wrong orientation by hydrogen bonds to ßGlu109 and the charged e-amino
group of ßLys87.
A second goal is to extend the successes in characterizing enzymatic transformations in PLP-dependent
enzymes to the ß-lactamases, starting with the Toho-1 ß-lactamase. Here we build on our initial chemical shift
assignments and characterization of dynamics in solution to study the chemical mechanism used to inhibit
antibiotics. In this application, NMR-assisted crystallography will be developed at the interface with neutron
crystallography, which to date has been unable to solve the structure in the presence of an inhibitor, but where
understanding the mechanism at the chemical level requires that we assign the protonation states of the key
active site acid/base catalytic residues.