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Shemyakin Institute of Bioorganic Chemistry, USSR
Academy of Sciences, Moscow, USSR.
Yuri A. Ovchinnikov started his carrier within the precincts of
Moscow University at the Chemical Department under Professor Yu.
A. Arbuzov. The project of his masters' degree (1957) provided material
for the first publication on a new technique for the synthesis of
pyrrolidine and thiophan derivatives. By that time, the gifted student
had already shown a disposition toward synthetic organic chemistry.
It was at this period that his belief took shape that the chemistry
of living organisms was by far the most attractive area for an organic
chemist to enter. Therefore, having begun his postgraduate course
at the Chemical Department, Y. A. Ovchinnikov readily accepted an
invitation to participate in the project on the complete synthesis
of an important group of antibiotics, the tetracyclines. While working
toward his doctorate, Yuri Ovchinnikov met M. M. Shemyakin, the
leader of the project. The joint work led to a long-lasting collaboration
between the two scientists, whose contribution to the foundation
and advancement of physicochemical biology in the USSR was outstanding.
After finished his postgraduate course, Ovchinnikov joined the Institute
for Chemistry of Natural Products of the USSR Academy of Sciences,
set up not long ago. Here, Professor Shemyakin proposed that he
go into peptide chemistry. The subject under study depsipeptide
antibiotics, atypical peptides containing hydroxy and amino acid
residues. The problems of synthesis of the optically active N-methylated
amino acids, reversible protection of the hydroxyl function of hydroxy
acids, and cyclization of linear depsipeptides were rapidly solved
and compounds with the structures proposed in the 1940s by Swiss
researchers for antibiotics enniatins A and B were prepared. However,
the samples obtained were devoid of antimicrobial activity and their
physicochemical properties differed much from those of the naturally
reliably confirmed, it remained to conclude that the formulae proposed
for enniatins A and B were incorrect. Several alternative structures
differing in ring size were suggested and accordingly synthesized.
Two of them were indistinguishable from the natural enniatins A
and B, which meant a solution to structural problems. Later (1964-1970),
Ovchinnikov and his colleagues performed a series of elegant syntheses
of some other naturally occurring depsipeptides (sporidesmolides
I-IV, angolidc, serratamolide, esperin, beauvericin). he was awarded
a D.Sc. in 1966 for the synthesis of natural depsipeptides and their
analogs. In 1967, Shemyakin, Ovchinnikov, and their team formulated
the original (socalled topochemical) principle of transformation
of biologically active peptides: novel molecules can be designed
by such deep structural modifications as reversal of the acylation
direction and the configuration of asymmetric centers, replacement
of ester bonds by amide bonds and vica versa, cyclization of linear
molecules, etc. The conditions favorable for retaining the original
stereo electronic parameters and, consequently, biological properties
of the molecule were found. Ideas from this pioneer research were
taken up by many laboratories and served to create novel highly
active peptides (hormones, antibiotics, neuropeptides, enzyme substrates,
and inhibitors). The experience accumulated during this synthetic
work served as a basis for the next and the culiminative step in
studying the depsipeptide antibiotics. Bearing in mind the recently
discovered ability of valinomycin and enniatins to induce permeability
of lipid membranes to alkali metals ions, Y. A. Ovchinnikov and
his colleagues undertook a study of the physicochemical basis of
the phenomenon. It appeared that valinomycin binds potassium ions
in solution, yielding stable complexes, and shows a unique K/Na-selectivily
of complex formation unsurpassed in nature. Enniatins bind virtually
all alkali and alkali-earth cations, though with a lower selectivity.
These complexes are the ion-transporting species, and selectivity
of ion binding is the origin of the selectivity of transmembrane
ion transport. Further, the threedimensional structures of the free
antibiotics and their complexes were established. It was shown for
the first time that such sophisticated structures can be resolved
not only by X-ray analysis but also in solution by spectral methods.
The bound ion appeared to reside always in the center of the depsipeptide
molecular cavity and be kept in place by ion-dipole interactions
with the carbonyl oxygens. The size of the valinomycin cavity is
limited by a bracelet-like system of six intramolecular hydrogen
bonds that accounts for its inability to adapt to smaller sized
ions such as sodium or lithium. Enniatin structures are more flexible,
which enables adjustment of the cavity to the size of the bound
ion. The molecular periphery of both valinomycin and enniatin complexes
is fully hydrophobic, which allows them to migrate freely across
lipid zones of the membrane. Several laboratories outside the USSR
were about to get similar results, but "the train had already gone."
Step-by-step protein compounds, the major working bodies of any
living system, began to occupy the prominent place in Ovchinnikov's
research activity. The 1970s witnessed a series of studies on the
primary structures of porcine aspartate aminotransferase, and toxins
from the venoms of cobra, bee, scorpion, etc. As a result, more
than 20 structures were added to international data banks and atlases
of protein structures. Inspired by these advances, Ovchinnikov and
his group tackled the deciphering of the primary structure of E.
coli DNA dependent RNA polymerase, a key transcription enzyme investigated
in many laboratories. Ovchinnikov had a very strong team, but even
for them the problem seemed extremely difficult, since RNA polymerase
is built of several subunits, among them two very large ß- and ß'-subunits
(each over 1300 amino acid residues). 1ndeed, after rapid sequencing
of the alfa-subunit (over 300 amino acids) it became clear that
analysis of the ß- and ß'-subunits exclusively by conventional methods
of protein chemistry could take many years. A decision was made
to utilize the methods of genetic engineering and to analyze the
sequences of genes coding for the subunits. In those days, such
an approach was new for this country, and elsewhere it was at the
early stages of development. Genes for large subunits of DNAdependent
RNA polymerase form the socalled operon rpo BC and contain about
10000 base pairs. They were isolated, inserted into plasmids, and
sequenced. Structures of pep tides of large subunits were detected
in parallel and independently. That was of use: when the structural
analysis of genes was completed and the structures of corresponding
proteins were derived according to the genetic code, they appeared
to coincide with the peptide structures and, consequently, were
determined correctly. Soon after that, other laboratories reported
the gene fragments but not the complete gene. It is worthwhile noting
that the structures of these fragments contained errors. only the
combined use of the methods of protein and nucleotide chemistry
provided reliable results. The structural analysis of RNA polymerase
served as a basis for a thorough investigation of the mechanism
of action of the enzyme, for numerous genetic and biochemical studies.
That was in the late 1970s. More and more laboratories outside the
USSR were successfully applying genetic engineering methods to microbiological
synthesis of practically important proteins. Yuri Ovchinnikov was
the first in the USSR to assess the prospects. He united enthusiasts
and headed the work on improving the methods of chemical synthesis
and directed mutagenesis of DNA to create microorganisms producing
alien peptides and proteins. As a result, strains producing an opioid
neuropeptide, leucine-enkephalin (1979), the antiviral and antitumour
human protein interferon-alfa2 (1981), and the precursor of human
insulin, proinsulin (1983), were obtained. Despite these advances
of Yuri Ovchinnikov in genetic engineering and biotechnology, the
bioorganic chemistry of peptides and proteins was always his major
interest and devotion. In the mid-1970s, he, N. Abdulaev, and a
group of colleagues focused their interest on the molecular mechanisms
of photoreception. By that time, a series of substantial discoveries
had been made that paved the way for solving the problem of how
light energy is transformed into the electric energy of the nerve
impulse by rhodopsin, a well-known light-sensitive protein from
the animal retina. Soon after wards, there appeared data on the
membrane protein -bacteriorhodopsin- found in microorganisms living
in salt lakes. The protein was given that name because of its similarity
to the visual rhodopsin (the presence of the bound retinal, light-sensitivity,
etc.). Though bacteriorhodopsin functioned as a light-dependent
proton pump, from the viewpoint of the primary photochemical properties
it was very similar to rhodopsin. At the same time, bacteriorhodopsin
is more readily available in large amounts and has a simpler structure
than the visual rhodopsin, the main effort was initially directed
to that protein. It was also considered that bacteriorhodopsin was
(and still is) an ideal model for structure-functional analysis
of membrane proteins. Simaltaneously with Prof. G. Khorana of the
USA, the Nobel prize winner, Ovchinnikov succeeded in determining
the amino acid sequence of bacteriorhodopsin, is was the first time
that the chemical structure of the membrane protein had been deciphered
(1987). Ovchinnikov and his team were then pioneers in solving the
structure of rhodopsin from bovine retina (1981). Research into
the topography of polypeptide chains of these proteins in native
membranes and elucidation of the structure of their active sites
and disposition of functionally important groups were the next steps
in this project. Using a variety of approaches including chemical
modification, enzymatic treatment, and immunochemical methods, Yuri
Ovchinnikov and his colleagues demonstrated that the two rhodopsins
are arranged in the membrane in a similar way as seven extended
protein segments spanning the membrane's width and connected with
each other on the two sides of the membrane by short peptide links.
In the mid-1980s, Y. Ovchinnikov and v. Lipkin focused their attention
on the studies of other proteins involved in transmission and amplification
of the visual cascade transducin and cyclic GMD phosphodiesterase.
In 1985, the primary structures of the y- and alfa-subunits of transducin
from bovine retinal rods were sequenced. Interestingly, the y-subunit
is characterized by the two adjoining cysteine residues also connected
by a disulfide bridge. The residues are apparently involved in the
formation of the transducin-photoactivated rhodopsin complex. An
exciting page in the scientific biography of Yuri Ovchinnikov was
his last project, devoted to studies of the system of active ion
transport, i.e., Na,Ktransporting adenosine triphosphatase and related
proteins. In the late 1970s, Ovchinnikov initiated research into
the structure of Na,K-ATPase. At the beginning, oligomeric organization
of the functionally active complex in the native membrane was unraveled
and the asymmetric arrangement of the subunits described. Further
progress depended upon determination of the amino acid sequence
of the subunits. Around 19851986, Ovchinnikov's team completed studies
of the nucleotide sequences of genes for subunits and amino acid
sequences of their polypeptide chains, which led to the complete
primary structure of Na,K-A TPase from pig kidney outer medulla.
Some research centers outside the USSR were also working intensively
in these areas. The teams of S. Numa (Japan) and A. Schwartz (USA)
simultaneously reported amino acid sequences of similar enzymes
from other sources. However, the approach chosen by Ovchinnikov
extended far beyond the primary structure determination. Complemented
by spectroscopic and molecular modelling studies, it resulted in
the first detailed model of the Na,K-ATPase spatial structure. Here,
the alfa-subunit (1016 amino acid residues) forms seven transmembrane
segments and the major portion of its hydrophilic region accommodating
the catalytic site is located inside the cell. The ß-subunit (302
residues) spans the membrane once and the main part of its polypeptide
chain forms an extracellular glycosylated domain. As for the Na,K-ATPase
active site, Ovchinnikov and his team employing affinity modification
by A TP analog succeeded in identifying an unknown component of
the catalytic site, thus experimentally confirming its dynamic changes
during enzyme functioning. Yuri Ovchinnikov, together with Eugene
Sverdlov and their groups of researchers, obtained novel data on
the regions of the human genome encoding the systems of active ion
transport that seem to be of general biological significance. A
family of at least five genes was defined in the human genome coding
for several isoforms of the Na,K-A TPase catalytic subunit as well
as other structurally similar ion-transporting A TPases. The discovery
of the multigene family gave rise to new concepts on regulation
of the active ion transport through changes in the activity of the
appropriate genes. This was supported by experiments on the expression
level of various genes for Na,K-ATPase in healthy and pathological
human tissues. Thus, ideas on the mechanisms of genetic regulation
of iontransporting enzymes received a solid foundation. Lately,
the problems of immunology and hematology attracted the attention
of Yuri Ovchinnikov, who believed that chemistry and biology should
do more to help solving medical problems in the USSR.Intense investigations
of naturally occurring regulators of immunity and hemopoiesis have
been started at the Shemyakin Institute. Some presentations at this
symposium deal with these problems. Above, we have outlined the
scientific interests of Yuri Ovchinnikov, who was also in the driving
seat in leading the chemical and biological scientific communities
of his country. Ovchinnikov could not imagine how the science could
evolve without intensive international cooperation. He excellently
presented the advances of the Institute, and promoted scientific
contacts, giving impetus to a series of bilateral symposia such
as USSR-FRG, USSR-USA, France-USSR, Sweden- USSR, and Italy-USSR
in various fields of physicochemical biology, many of which have
now became a tradition. The remarkable symposia on Frontiers in
Bioorganic Chemistry and Molecular Biology in Tashkent (1980) and
Moscow-Alma-Ata (1984) were also organized and presided over by
him. Of Yuri Ovchinnikov occupies a prominent place in the world's
scientific heritage.
We can only guess at what his further endeavors would have been,
if he were still alive. It is our hope that this numerous works
will inspire many generations of bioorganic chemists to come, providing
the key to solving a diversity of problems and demonstrating again
and again the beauty and the attractive power of the world of science.
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