Crystallography of enzymes
J. Dohnálek1,2, T. Skálová1, T.
Kovaľ2, J. Dušková1, P. Kolenko2, K. Fejfarová2,
J. Stránský1, L. Švecová1, M. Trundová1, J.
Hašek1,2
1Institute of Biotechnology, v. v. i. AS CR, Vídeňská 1083, 14220 Praha 4
2Institute of Macromolecular Chemistry, v. v. i., AS CR, Heyrovského nám. 2, 16206 Praha 6
dohnalek@ibt.cas.cz
Knowledge
of three-dimensional structure of enzymes plays an important role in our
understanding their function and is required for design and modification of
enzymatic properties and stability. In recent years we have worked with several
enzymatic systems where structural studies provided very important insights
into the enzyme behaviour, its classification, and
helped discovery of previously not known activity or uncovered structural
changes upon ligand binding.
In
structural studies of β-galactosidase from Arthrobacter sp. C2-2 a unique way of arrangement of
the molecules in functional hexamers was discovered.
The functional form of the enzyme revealed by its crystal structure has
consequences for the actual substrate and product logistics. The sphere-like hexamers form a large cavity inside the cluster with three
types of channels connecting it with exterior. The six active sites of the hexamer are open into the internal cavity and are not
accessible from the outside. Thus any substrate or ligand
must pass through the channels of the hexamer to
reach the catalytic site. In further studies it was confirmed that ligands can indeed access the active sites inside the
cavity via the existing channels. Also presence of the so called shallow
binding mode of this glycosyl hydrolase
was confirmed by observation of inhibitor binding in this 660 kDa structure [1].
Structural
arrangement and stabilization features uncovered by crystal structure of the
small laccase from Streptomyces coelicolor for the first time proved
existence of the trimerization-dependent laccase, in which quaternary organization in an interesting
way makes the second domain of laccase redundant and
ensures extreme stability [2]. Here, an exceptionally high solvent content
value of 83% was observed. The high solvent content was interesting from the
point of view of methodology of structure solution as it enabled very effective
application of solvent flattening to the initial phases acquired from a MAD
experiment on copper ions. Further studies of ligand
binding led to variation of enzyme arrangement in the crystal and improved
quality of data connected with ferrocyanide binding
(acting as an electron donor). The structure solution revealed also presence of
a central channel which can serve as a route for access to the trinuclear copper cluster. Its role still remains
unexplained.
Structure
of the plant nuclease TBN1 with confirmed anticancerogenic
properties opened up many topics regarding non-specificity of this enzyme
capable of degradation of ss and ds
DNA and RNA. Structural similarity to another enzyme led to confirmation of phospholipase C-like activity and initiated other
investigations [3]. Especially mapping of the molecular surface electrostatics
and differences between single strand and double strand processing nucleases of
this type suggest some amino acid residues being responsible for such
specificity. Structural studies also brought insights into aggregation/dimer formation of this enzyme, which for the first time
raises questions about specific peptide binding in a nuclease active site.
Structural
study of a bacterial organophosphorous acid anhydrolase provided for the first time a complete view of
the enzyme and proved that the enzyme classes of prolidase
and OPAA are basically identical [4]. Access to the active site differs in the
human and bacterial enzymes although dependence on manganese ions remains
conserved. The solved structures showed details important for enzyme dimerization, which is required for function. Covalent
modification of the peptide chain of the bacterial OPAA was observed, which was
identified as nickel forming covalent bonds to the main chain nitrogen atoms.
X-diffraction
analysis of enzymes brings essential information directly related to function
of the studied systems. The most outstanding are information about oligomerization and its exact mechanism, positioning and
exact function of the catalytic amino acids, ligand
and metal binding, and mechanisms of structural stabilization. Solid results of
structural analysis pave route to further modifications of the enzymatic
systems and utilization in biotechnological and medical applications.
Acknowledgement: Support is acknowledged from the Czech Science Foundation (no. P302/11/0855), MEYS (LG14009, EE2.3.30.0029) and BIOCEV
CZ.1.05/1.1.00/02.0109 from the ERDF.
References
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