A4 I8 Biochemical Society Transactions (2000) Volume 28, Part 5
1516 Interaction of a hydrophobic ligand with the FAD-binding
site of glucose oxidase
M.Nemat-Gorgani
Institute of Biochemistry and Biophysics, University of Tehran,
Tehran 13141-1384, Iran
This investigation involved use of palmityl-substituted Sepharose4B, a hydrophobic support previously found to immobilize a
number of proteins with retention of native properties. Some of
these proteins have been found to adsorb on this matrix either
under normal conditions or in the course of reversible denaturation process. During this studies, it was observed that glucose
oxidase which is not normally immobilized on this support gains
such capacity by thermal denaturation. The extent of immobilization of the apoenzyme after and before incubation with FAD
showed that the alkyl chains compete with FAD for binding to
the apoprotein. It therefore appears that interaction of apoenzyme
of glucose oxidase with matrices substituted with hydrophobic
ligands may provide useful information on the hydrophobic
pockets of the proteins in which FAD is accomodated. It is also
suggested that binding of the palmityl residues to the FAD site
may stabilize the three-dimensional structure of the protein in the
same manner as that shown by FAD and ADP. The approach may
be utilized for other flavoproteins or nucleotide-requiring
enzymes.
1518 Proteolysis of Mesophilic and Thermophilic a Amylases; A
Comparative Study
K. Khajeh, M . Nemat-Gorgani
Institute of Biochemistry & Biophysics, University of Tehran,
Tehran,13141-1384,Iran
In this investigation, a mesophilic a amylase obtained from
Bacillus amyloliquefaciens (BAA) was compared with a thermophilic a amylase obtained from Bacillus licheniformis (BLA) in
relation to limited and extensive proteolysis by trypsin. The
digestion patterns are explained in terms of available information
in the literature on Structure of these proteins, especially in relation to segmental mobility. While the catalytic potential of BLA
was enhanced upon proteolysis, that of BAA was diminished due
to this process. Combined with greater catalytic activity, a lower
thermal stability was observed for BLA upon proteolytic treatment. Results presented in this comparative study indicate a higher resistance of the thermophilic enzyme toward digestion by
trypsin, evidently due to a greater degree of Structural rigidity of
this protein. The limited digestion patterns obtained are in accordance with suggestions in the literature on positioning of flexible
areas (loops) in the protein StruCNreS which constitute the primary targets for proteolytic attacks. For both enzymes, various
additives have been shown to diminish proteolysis similar to their
protection against other unfavourable conditions described in a
separate investigation reported recently.
1517 Rational approaches to the homology modelling of 3D struc1519 Crystal Structure of new class Rubisco from Thermococcus
NreS of cytochromes P450.
A
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S
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A.V. Dubanov, A.A.
Sechenykh, V.S.
Skvortsov, A.I. Archakov
Inst. Biomed. Chem., Pogodinrkaya stx 10, Moscow, 119832, Russia
Cytochromes P450 (P450) play a key role in xenobiotics oxidation.
Insufficient number of known 3D structures of P450s forces the scientists to use computer molecular modelling. However this
approach meets significant difficulties stipulated by low similarity
of P450s sequences. The main problems are: 1) disagreement
between optimal text and 3D alignments of template Structures; 2) it
is extremely difficult to obtain optimal 3D alignment; 3) too short
SCRs (structure conserved regions) poses great difficulties in determination of appropriate sites in modeled protein; 4) too long loops
is a problem for conformation searching; 5) designed model requires
deep optimization, that is also nontrivial problem.
In present work we have executed Swiss-Prot database mining with
the help of the original method sequentially used local and global
text alignments and the sequences of P450s with known 3D structures as the requests. The list of P450s, which can be successfully
modeled based on homology, was composed.
The alternate homology modelling of all P450s with known 3D
structures were performed. The obtained models were compared
with known x-ray structures. The outcomes of this test analysis
were compared with results produced by special verification software. Some rules for modelling of P450 with relatively low homology were formulated and applied for P45011A1 modelling. This
model was successfully validated by special software verification
and known indirect experimental data.
This work was supported in part by RFBR grant 99-04-48754.
We thank Tripos GmbH (Munich, Germany) for scientific and technical support.
0 2000 Biochemical Society
kodukaraensis KODl with pentagonal symmetry
N. Maeda, H.Atomi, T.Imanaka, K. Miki
Graduate School of Science, Kyoto University, Sakyo-ku, Kyoto
606-8102,Japan, and Graduate School of Engineering, Kyoto
University, Sakyo-ku, Kyoto 606-8101,Japan
Ribulose-1,5-bisphosphatecarboxylase/oxygenase (Rubisco) catalyzes the primary step of COq fixation which is located at the
origin of photosynthetic dark-reactions. This enzyme catalyzes
the covalent addition of C 0 2 to ribulose 1,5-bisphosphate producing two molecules of 3-phosphoglycerate. Rubiscos were so
far classified fundamentally into two types, Type I from plants
and Type I1 from bacteria. Type I is composed of eight large subunits and eight small subunits (L&) with local 4-fold axis, but
Type I1 is composed only of two large subunits (L2). Recently,
highly active Rubisco in a hyperthermophilic archaeon,
Thennococcus (formerly Py~ococcus)kodukaraensis KODl (TKRubisco), were characterized. Phylogenetic analysis indicated that
archaeal Rubisco (Type 111),including Tk-Rubisco, were different
from previously reported Type I or Type I1 Rubiscos in terms of
primary structure. Tk-Rubisco was crystallized using ammonium
sulfate as precipitant. The crystals belong to the space group
P3121 with the cell dimensions of a = 233.7A and c = 93.2A. The
crystal structure of Tk-Rubisco determined at 2.8A resolution
(current R = 23.8%, Rfree = 27.2%). showed that this enzyme is
the (L2)5 pentagonal ring-like decamer composed only of large
subunits. This is the first report of the decameric structure of
Rubisco, which should be classified into a new type (Type 111).