Biophysics and Molecbsuular Biology

Bacillus pasteurii UreE (BpUreE) is a putative chap-
erone assisting the insertion of Ni
2
ions in the active
site of urease. The x-ray structure of the protein has
been determined for two crystal forms, at 1.7 and 1.85 Å
resolution, using SIRAS phases derived from a Hg2
-
derivative. BpUreE is composed of distinct N- and C-
terminal domains, connected by a short flexible linker.
The structure reveals the topology of an elongated ho-
modimer, formed by interaction of the two C-terminal
domains through hydrophobic interactions. A single
Zn2
ion bound to four conserved His-100 residues, one
from each monomer, connects two dimers resulting in a
tetrameric BpUreE known to be formed in concentrated
solutions. The Zn2
ion can be replaced by Ni
2
as shown
by anomalous difference maps obtained on a crystal of
BpUreE soaked in a solution containing NiCl2. A large
hydrophobic patch surrounding the metal ion site is
surface-exposed in the biologically relevant dimer. The
BpUreE structure represents the first for this class of
proteins and suggests a possible role for UreE in the
urease nickel-center assembly.

Using affinity chromatography on 8-(2-aminoethyl)-amino-cAMP Sepharose, the cGMP-dependent protein kinase (ATP: protein phosphotransferase, EC 2.7.1.37) from tissues of the prawn Palaemon adspersus was purified to homogeneity as demonstrated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The degree of enzyme purification was 11 200, recovery--6.5%; the isoelectric point for the enzyme lies at 5.5. Data from gel filtration and centrifugation in sucrose density gradient suggest that the dimer of cGMP-dependent protein kinase has a molecular weight of 157 000, sedimentation coefficient of 7.2S and a Stokes' radius of 50 A. An active form of the enzyme with Mr = 76 500 (4.5S, 39 A) which apparently represents a subunit of the cGMP-dependent protein kinase was discovered. The activity of the both enzyme forms are stimulated by low concentrations of cGMP (Ka = 1.10(-7) M). The monomer and dimer molecules appear as prolate ellipsoids with axial ratios ...

Using ion-exchange chromatography and gel filtration, cGMP-dependent protein kinase was purified from prawn tissues 220-fold with a yield of activity of 12%. The apparent Ka values for cGMP, cAMP and 8-Br-cGMP are 1 . 10(-7), 5 . 10(-6) and 5 . 10(-8) M, respectively; the apparent Km values for ATP in the presence of cGMP is 9 . 10(-6) M. The cGMP-stimulated protein kinase activity was observed only in the presence of SH-compounds and high Mg2+ concentrations (500-100 mM). The protein kinase demonstrated a broad pH optimum wih a maximum at pH 6.8-7.2. The elution volume of the enzyme during gel filtration corresponded to a globular protein with molecular weight of 140,000.

Porphyrin nanotubes have been obtained from tetrakis (4-sulfonatophenil) porphyrin (TTPS4) using ionic self-assembly, and UV-vis spectra have been recorded. pH titration of the Soret band has been performed at 420 nm. It has been shown that by decreasing pH from pH 7.7 to pH 1.0 the changes in UV-vis spectra characteristic for porphyrin aggregation (batochromic shift) take place. At pH 7.7 in TPPS4 spectra the peaks at 420 (Soret band) and at 555, 596 nm (Q bands) are observed. At pH 1.0 the peak of the Soret band shifts to 434, whereas Q bands shift to 589 and 644 nm, respectively. It has been found that Autodock Vina program allows to create realistic 2D structural models of porphyrin monolayers adsorbed on a graphene substrate. After the first layer is completely formed owing to pi-pi iteractions and van der Waals forces between porphyrin and graphene the second and consequent layers begin to form due to porphyrin J- and H-aggregations.

The paper deals with the mechanism of the phosphotransferase reaction catalyzed by G-kinase purified from the shrimp tissue by affinity chromatography. The ATP functional groups are stated to be significant for interaction with the enzyme. A mathematical model of the reaction is suggested assuming two active centres in a G-kinase molecule.

Selective Escherichia coli β-glucuronidase (eGUS) inhibitors, which do not affect human β-
glucuronidase (hGUS), greatly alleviate side effects (severe diarrhea) of worldwide used chemotherapeutics,
camptothecin and its derivatives. We have studied the probable mechanism of the selectivity of eGUS inhibition.
Structural superposition has shown high structural homology in tertiary structure of hGUS and eGUS. Active
center models of the enzymes have been build, and the principal difference in quaternary structure of the two
enzymes has been found. The four active centers of the human enzyme are positioned separately, each in the area
of its own polypeptide chain; their amino acids are positioned distantly from those of the neighboring subunits.
In contrast, each eGUS active center includes Phe365 amino acid residue derived from a neighboring polypeptide
chain. The improved model of eGUS subunit substrate binding site suggests that future eGUS molecular docking
and structure based in silico virtual screening studies should be performed with the dimeric structure rather than
with a single subunit. All presently known natural eGUS inhibitors have been characterized, the possibility and
expediency of obtaining of such inhibitors from herbal sources has been justified. The part of the research is
aimed to advance towards “green” technologies in pharmaceutics.

UreE is a homodimeric metallo-chaperone that assists the insertion of Ni(2+) ions in the active site of urease. The crystal structures of UreE from Bacillus pasteurii and Klebsiella aerogenes have been determined, but the details of the nickel-binding site were not elucidated due to solid-state effects that caused disorder in a key portion of the protein. A complementary approach to this problem is described here. Titrations of wild-type Bacillus pasteurii UreE (BpUreE) with Ni(2+), followed by metal ion quantitative analysis using inductively coupled plasma optical emission spectrometry (ICP-OES), established the binding of 2 Ni(2+) ions to the functional dimer, with an overall dissociation constant K(D) = 35 microM. To establish the nature, the number, and the geometry of the ligands around the Ni(2+) ions in BpUreE-Ni(2), X-ray absorption spectroscopy data were collected and analyzed using an approach that combines ab initio extended X-ray absorption fine structure (EXAFS) calculations with a systematic search of several possible coordination geometries, using the Simplex algorithm. This analysis indicated the presence of Ni(2+) ions in octahedral coordination geometry and an average of two histidine residues and four O/N ligands bound to each metal ion. The fit improved significantly with the incorporation, in the model, of a Ni-O-Ni moiety, suggesting the presence of a hydroxide-bridged dinuclear cluster in the Ni-loaded BpUreE. These results were interpreted using two possible models. One model involves the presence of two identical metal sites binding Ni(2+) with negative cooperativity, with each metal ion bound to the conserved His(100) as well as to either His(145) or His(147) from each monomer, residues found largely conserved at the C-terminal. The alternative model comprises the presence of two different binding sites featuring different affinity for Ni(2+). This latter model would involve the presence of a dinuclear metallic core, with one Ni(2+) ion bound to one His(100) from each monomer, and the second Ni(2+) ion bound to a pair of either His(145) or His(147). The arguments in favor of one model as compared to the other are discussed on the basis of the available biochemical data.