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Plant Physiol, January 2001, Vol. 125, pp. 54-57 Plant Enzyme Structure. Explaining Substrate Specificity and the Evolution of FunctionDepartment of Plant Science, University of Adelaide, Waite Campus, Glen Osmond, South Australia 5064, Australia
Progress in defining the
three-dimensional (3D) structures of plant enzymes has been generally
slow, but in the last 5 years momentum has picked up considerably (Fig.
1). By the beginning of 2000 about 140 individual plant protein structures were known, of which 37 related to
individual plant enzymes.
Most 3D structural data have been generated by x-ray crystallography. The first and very often the limiting step in this procedure is the production of enzyme crystals. However, if high-quality crystals can be obtained, solving the 3D structure can be greatly facilitated by the recent advent of more powerful x-ray generators, such as synchrotrons coupled with multiwavelength anomalous diffraction, increased computing power, and the use of molecular cloning for rapid determination of amino acid sequences. But where has all this led us? The emerging conclusion is that both prokaryotic and eukaryotic proteins are comprised of an unexpectedly small number of protein folds, which can be combined, adapted, and fine-tuned to achieve the diverse and quite specific functions mediated by the very large number of proteins that operate at the cellular level. For example, domains that mediate protein-protein interactions are conserved in plant and animal proteins that range in function from regulators of transcription and cytoskeleton organization, to proteins that form K+ channels across membranes (1). Thus, a relatively small number of structural elements has been conserved, but these are used over and over again in the diversification of protein function during evolution. This is also the case for plant enzymes, in which 3D structure ultimately defines substrate specificity and therefore function.
As more 3D structures are solved by x-ray crystallography and NMR, it is becoming apparent that proteins with 25% to 30% sequence identity over 100 or more amino acid residues are likely to have similar 3D conformations. If the 3D structure of one such protein is known, the structure of the other can be deduced by homology modeling (9). Structures obtained by modeling will be less reliable than those determined experimentally by x-ray crystallography, but can nevertheless provide valuable information on enzyme fold and function. For example, Harvey et al. (3) used homology modeling to examine the 3D
structures of enzymes in the family 3 group of glycoside hydrolases.
The only member of the family for which a 3D structure was available
was the In another seminal example, homology modeling allowed the 3D structure of the bean storage protein phaseolin to be linked with the structures of the large group of enzymic and nonenzymic proteins that constitutes the cupin superfamily (2). Our understanding of the evolution of plant enzymes is likely to be greatly enhanced by these structural "connections." Finally, automated 3D structural modeling programs can be used to rapidly identify unknown proteins and enzymes in high-throughput genomics programs. In this procedure, a "structure" constructed by modeling the amino acid sequence of an unknown protein is compared with actual 3D structures in the databases, using increasingly powerful computers and analytical algorithms. The method has been applied with considerable success in yeast genome projects for the identification of genes encoding unknown proteins (10).
The fundamental factors that determine substrate specificity of enzymes are conformational and chemical complementarity between the substrate and its binding site on the enzyme. Thus, the binding site usually consists of a cleft, tunnel, funnel, or other depression on the enzyme's surface. Only those substrates that have complementary shapes will fit into the binding site. Perhaps most intriguing from an evolutionary viewpoint is the precise alignment, or chemical complementarity, of interactive amino acid side-chains on the enzyme surface with corresponding groups on the substrate. Some general rules of substrate specificity are emerging, in particular for groups of enzymes with common action patterns. For example, an endohydrolase usually has a substrate binding groove or depression that extends across its surface, whether it is a polysaccharide, nucleic acid, or polypeptide endohydrolase (Fig. 2A). Catalytic amino acid residues are located in the substrate-binding cleft. As a result, the endohydrolase can essentially bind anywhere along the polymeric substrate and hydrolyze internal linkages.
In contrast, an exohydrolase needs to align its substrate such that
terminal linkages are juxtaposed to catalytic residues. This is usually
achieved through a dead-end tunnel, slot, or funnel in the enzyme (Fig.
2, B and C). Specificity can be adjusted from "tight" or
"loose" depending on the dimensions of the tunnel or on the
geometry of the substrate-binding site. A deep, narrow funnel of the
kind observed for barley The tight specificity of the barley
Higher plants synthesize a range of enzyme inhibitors that
function by binding into the active site of the target enzyme, thereby
preventing the approach of the natural substrate. As with substrate
binding, inhibitor binding requires elements of shape and chemical
complementarity between the inhibitor and the enzyme. The 3D structures
of a number of enzyme-inhibitor complexes are now solved and provide
detailed information on inhibitor action and specificity. In the case
of plant
The evolution of enzymic activity and specificity can follow two
very different routes (7). First, a protein without catalytic activity
but with some well-developed binding capacity for a particular metabolite might accumulate mutations until catalysis occurs. As
mentioned earlier, an evolutionary link between enzymic and nonenzymic
proteins in the cupin superfamily has been suggested by 3D structural
studies and molecular modeling (2). The cupin domain consists of two
conserved motifs, each of about 20 amino acids in length and connected
by a linker peptide of variable length, which form small The second evolutionary route to enzyme activity and specificity occurs
when an enzyme capable of performing the required catalysis undergoes
mutational changes that result in altered substrate specificity.
An example here was provided by x-ray crystallography of barley
(1
What new concepts have developed from 3D structural studies of plant enzymes and how might these contribute to our future understanding of plant physiology? We can confidently predict that 3D structural analyses of the type used to define enzyme-substrate interactions and the mechanisms of enzymatic catalysis will be extended more broadly into studies on plant cell biology and that molecular modeling will continue to play an important role in these studies. Protein/ligand interactions, other that those of the enzyme/substrate type, will be described in 3D detail. For example, protein inhibitor/enzyme binding, docking of phytohormones with their receptors, the action of specific transporter proteins, and the binding of transcription factors to specific nucleotide sequence motifs could be accurately defined through 3D studies. Protein/protein interactions that encompass such fundamental processes as signal transduction and plant-pathogen interactions could also be understood through x-ray crystallography. Developing technologies will need to address problems associated with obtaining 3D structures of membrane-bound proteins and in describing real-time changes in protein conformations during their interactions with other molecules. Time-resolved crystallography, neutron crystallography, and electron cryo-crystallography offer considerable promise in these areas. If these types of protein/protein and protein/ligand interactions in plants can be described in precise molecular and 3D structural terms, we will place ourselves in a strong position to truly understand the central processes of cell biology. Further, this understanding will present opportunities to enhance plant productivity and end-product quality through rational modification (4) or directed molecular evolution (13) of existing enzymes and de novo design of catalytic proteins. Additional applications in plant production could include the use of specific enzyme inhibitors to regulate or manipulate processes of growth and development.
We gratefully acknowledge grants obtained from the Grains Research and Development Corporation and the Australian Research Council. We also thank Andrew Harvey for his assistance with the figures and Dr. Jose Varghese for invaluable discussions.
* Corresponding author; e-mail geoffrey.fincher{at}adelaide.edu.au; fax 61-8-8303-7109.
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Three-Dimensional Structures of...
Molecular Modeling
-glucan exohydrolase from barley (12). However, this single
structure could be used to build reliable models of most of the 100 or
so members that constitute this family of enzymes, even though sequence
identities of members were as low as 22%. The modeling also revealed
that enzymes could be constructed through circular permutations of
domains; the sequence of protein domains can be altered without
affecting the final 3D structure of the entire enzyme. As a result of
the modeling exercise, numerous research groups working on family 3 enzymes from bacteria, fungi, and higher plants have been provided with
structural clues as to substrate specificity and catalytic mechanisms
of their enzymes of interest. Further, biological function can be
predicted with a higher degree of confidence.
3,1
-amylase inhibitors, the 3D structures provide the
molecular detail to explain why inhibitors specifically inhibit
exogenous 
