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Copyright 2008
University of Florida
Questions or comments about this site should be sent to Captain Jack
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Nigel Richards
 Professor of Chemistry
B.Sc., University of London, 1980 (Chemistry)
Ph.D., Cambridge University, 1983
Harkness Fellow, Columbia University, 1983-1985
richards@qtp.ufl.edu
428 Leigh Hall
(352) 392-3601
Enzyme structure and mechanism;
Evolution of enzyme activity; Manganese-dependent enzymes; Glutamine-dependent amidotransferases;
Computational enzymology; Chemical biology & drug discovery
Enzyme Evolution and Oxalate Metabolism
Oxalic acid is produced in
large quantities by cellular metabolism, and a number of pathological
conditions can arise if oxalate accumulates in Man. These include hyperoxaluria, the
formation of calcium oxalate stones in the kidney (urolithiasis), renal
failure, cardiomyopathy and cardiac conductance disorders. There are no
drug-based therapies for these conditions, and so there is a lot of interest
in studying oxalate metabolism and the enzymes that are involved in the
biosynthesis and degradation of oxalate. A number of enzymes have evolved in
plants (oxalate oxidase), fungi (oxalate decarboxylase) and bacteria
(oxalyl-CoA decarboxylase/formyl-CoA:oxalate transferase) that degrade
oxalate 1 to formate 2 and carbon dioxide:
The observation that these
three groups of enzymes appear to utilize very different mechanisms to
degrade oxalate, a kinetically difficult chemical transformation, is of
catalytic and evolutionary interest. Our group is therefore carrying out
structural, spectroscopic and kinetic studies of these enzymes in order to
understand (i) the mechanisms by which oxalate is degraded, and (ii) the
underlying processes of molecular evolution that have given rise to these
protein catalysts.
Glutamine-Dependent Amidotransferases
 Asparagine synthetase is a glutamine-dependent, Ntn amidotransferase
that mediates the cellular biosynthesis of L-asparagine from aspartic acid. Several
lines of evidence suggest that inhibitors of the asparagine synthetase have
potential clinical application in the treatment of leukemia and solid tumors.
Our group is therefore carrying out multi-disciplinary studies into the
structure and mechanism of this enzyme. These experiments involve the
synthesis of novel analogs of enzyme substrates and reaction intermediates,
and their characterization as inhibitors of the enzyme. Our recent structural
studies have also shown that asparagine synthetase contains an intramolecular
channel through which ammonia, released by glutamine hydrolysis in the
N-terminal active site, is transferred to a nitrogen acceptor formed in a
second, C-terminal active site. We are therefore using site-directed
mutagenesis and directed evolution methods to investigate the molecular
mechanisms that have been employed to construct this substrate channel. These
studies are also likely to yield information of general scientific importance
to efforts to develop novel catalytic activities by the preparation of hybrid
enzymes. [The figure on the left shows a representation of the crystal
structure of Escherichia coli asparagine synthetase B, in which the
protein atoms are rendered in green, and bound glutamine and AMP located in
the two active sites are rendered in standard CPK atom colors.]
Computational Bioinorganic Enzymology
The role of
transition metals in enzyme catalysis and the modulation of metal reactivity
by the protein environment are questions that are often difficult to
investigate experimentally. With the advent of density functional theory
(DFT), however, accurate calculations of the electronic structure and
reactivity of transition metal-containing enzymes have become possible. Our
group is therefore applying these computational methods to determining the
molecular properties of Fe- and Mn-dependent metalloenzymes. As part of these
efforts, we are studying the unique non-heme Fe(III) center in nitrile
hydratase. Many important structural and mechanistic details concerning
the role of the metal in catalysis and regulation of the enzyme by nitric
oxide remain poorly understood. These include the relationship between spin
state preference and protein structure, and the observation that
post-translational oxidation is essential for biological activity. In this
project, DFT calculations are being used to determine the molecular basis for
the unexpected spin preference of the metal center, and the role of
post-translational modification in allowing the enzyme to catalyze the
hydration of nitriles to primary amides. In addition, new DFT/MM techniques
are being employed to characterize the photochemical properties of the
Fe(III) center in the enzyme, and their role in regulating the activity of
nitrile hydratase. [The figure shows the high-resolution crystal structure of
the Fe(III) center in the inactive form of nitrile hydratase. In this
structure, NO is bound to the metal in the sixth coordination site. Atom
coloring: C – grey; N – blue; O – oxygen; S – yellow;
Fe – orange.]
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