Zsolt Böcskei

Prolyl Oligopeptidase: An Unusual β-Propeller Domain Regulates Proteolysis

Abstract Prolyl oligopeptidase is a large cytosolic enzyme that belongs to a new class of serine peptidases. The enzyme is involved in the maturation and degradation of peptide hormones and neuropeptides, which relate to the induction of amnesia. The 1.4 A resolution crystal structure is presented here. The enzyme contains a peptidase domain with an α/β hydrolase fold, and its catalytic triad (Ser554, His680, Asp641) is covered by the central tunnel of an unusual β propeller. This domain makes prolyl oligopeptidase an oligopeptidase by excluding large structured peptides from the active site. In this way, the propeller protects larger peptides and proteins from proteolysis in the cytosol. The structure is also obtained with a transition state inhibitor, which may facilitate drug design to treat memory disorders.

Phospholes with reduced pyramidal character from steric crowding III NMR and X-ray diffraction studies on 1-(2,4,6-tri-isopropylphenyl)-3-methylphosphole

Abstract The 2,4,6-tri-isopropylphenyl substituent was placed on the phosphorus of a phosphole to reduce the pyramidal character. That this was accomplished was revealed by single crystal X-ray diffraction analysis; with respect to the plane of C2PC5 in the phosphole ring, the ipso carbon of the benzene ring was deflected by only 58.0°, whereas the deflection is 66.9° in the uncrowded 1-benzylphosphole. This proves that the concept of reducing the pyramidal character (with the goal of increasing the electron delocalization) through steric crowding can be realized. In the crystal the two rings are in orthogonal planes, but this relation is not retained in solution; NMR studies show that the two edges of the benzene ring, as well as the 2,6-isopropyl groups, are identical.

Synthesis, absolute configuration and intermediates of 9-fluoro- 6,7- dihydro-5-methyl-1-oxo-1H,5H-benzo[i.j]quinolizine-2-carboxylic acid (flumequine)

Abstract The antibacterial agent 9-fluoro-6,7-dihydro-5-methyl-1-oxo-1H,5H-benzo[i,j]quinolizine-2-carboxylic acid (flumequine) was synthesized in optically active form from 6-fluoro-2-methyl-1,2,3,4-tetrahydroquinoline (FTHQ). Racemic FTHQ was resolved with the enantiomers of 3-bromocamphor-8-sulfonic acid. The configurations were established by X-ray structures of the two diastereoisomeric salts. Enantiomeric excesses were determined by 1H NMR analysis.

Efficient synthesis and resolution of (±)-1-[2-carboxy-6-(trifluoromethyl)phenyl]pyrrole-2-carboxylic acid

Abstract A novel, efficient synthesis and resolution of (±)-1-[2-carboxy-6-(trifluoromethyl)-phenyl]pyrrole-2-carboxylic acid has been developed for the preparation of new members of optically active atropisomers. The e.e. values were determined by a highly sensitive 19F NMR spectroscopic method using β-cyclodextrin as chiral complexing agent. Single-crystal X-ray structures of the two diastereoisomeric salts and consequently, the absolute configurations of the enantiomers are also reported.

The ability of 2-phosphabicyclo[2.2.2]oct-5-ene 2-oxides to undergo fragmentation of the bridging P-moiety

Abstract New 2-phosphabicyclo[2.2.2]octene 2-oxides (2 and 3) were synthesized by the Diels–Alder reaction of 1,2-dihydrophosphinine oxides (1) and dienophiles, such as N-phenyl maleimide and maleic anhydride. The X-ray structure of one of the products (2Ab) suggests that the phosphabicyclooctenes have a less strained framework than the phosphabicyclooctadienes described earlier. As a consequence of this, also confirmed by thermal examinations and semiempirical calculations, thermal fragmentation of the phosphabicyclooctenes requires more forcing conditions, than that of the bicyclooctadienes. The methylenephosphine oxide (10) ejected could be utilized in the phosphorylation of hydroquinone in moderate yield. Mass spectral fragmentation of cycloadducts 2 and 3 under electron-impact conditions seems to be in agreement with the preparative experiences.

Human Prorenin Structure Sheds Light on a Novel Mechanism of Its Autoinhibition and on Its Non-Proteolytic Activation by the (Pro)renin Receptor

Antibodies and prorenin mutants have long been used to structurally characterize prorenin, the inactive proenzyme form of renin. They were designed on the basis of homology models built using other aspartyl protease proenzyme structures since no structure was available for prorenin. Here, we present the first X-ray structure of a prorenin. The current structure of prorenin reveals that, in this zymogene, the active site of renin is blocked by the N-terminal residues of the mature version of the renin molecule, which are, in turn, covered by an Ω-shaped prosegment. This prevents access of substrates to the active site. The departure of the prosegment on activation induces an important global conformational change in the mature renin molecule with respect to prorenin: similar to other related enzymes such as pepsin or gastricsin, the segment that constitutes the N-terminal β-strand in renin is displaced from the renin active site by about 180° straight into the position that corresponds to the N-terminal β-strand of the prorenin prosegment. This way, the renin active site will become completely exposed and capable of carrying out its catalytic functions. A unique inactivation mechanism is also revealed, which does not make use of a lysine against the catalytic aspartates, probably in order to facilitate pH-independent activation [e.g., by the (pro)renin receptor].

Role of electrostatics at the catalytic metal binding site in xylose isomerase action: Ca2+‐inhibition and metal competence in the double mutant D254E/D256E

The catalytic metal binding site of xylose isomerase from Arthrobacter B3728 was modified by protein engineering to diminish the inhibitory effect of Ca2+ and to study the competence of metals on catalysis. To exclude Ca2+ from Site 2 a double mutant D254E/D256E was designed with reduced space available for binding. In order to elucidate structural consequences of the mutation the binary complex of the mutant with Mg2+ as well as ternary complexes with bivalent metal ions and the open‐chain inhibitor xylitol were crystallized for x‐ray studies. We determined the crystal structures of the ternary complexes containing Mg2+, Mn2+, and Ca2+ at 2.2 to 2.5 Å resolutions, and refined them to R factors of 16.3, 16.6, and 19.1, respectively. We found that all metals are liganded by both engineered glutamates as well as by atoms O1 and O2 of the inhibitor. The similarity of the coordination of Ca2+ to that of the cofactors as well as results with Be2+ weaken the assumption that geometry differences should account for the catalytic noncompetence of this ion. Kinetic results of the D254E/D256E mutant enzyme showed that the significant decrease in Ca2+ inhibition was accompanied by a similar reduction in the enzymatic activity. Qualitative argumentation, based on the protein electrostatic potential, indicates that the proximity of the negative side chains to the substrate significantly reduces the electrostatic stabilization of the transition state. Furthermore, due to the smaller size of the catalytic metal site, no water molecule, coordinating the metal, could be observed in ternary complexes of the double mutant. Consequently, the proton shuttle step in the overall mechanism should differ from that in the wild type. These effects can account for the observed decrease in catalytic efficiency of the D254E/D256E mutant enzyme. Proteins 28:183–193, 1997.

Microbial transformation of β-sitosterol and stigmasterol into 26-oxygenated derivatives

The genetically modified Mycobacterium sp. BCS 396 strain has been used to transform sterols with stigmastane side chain in order to obtain 26-oxidized metabolites. beta-Sitosterol (I) was transformed to 4-stigmasten-3-one (II), 26-hydroxy-4-stigmasten-3-one (III), and 3-oxo-4-stigmasten-26-oic acid (IV), while stigmasterol (V) was converted to 4,22-stigmastadien-3-one (VI), 6 beta-hydroxy-4,22-stigmastadien-3-one (VII), 26-hydroxy-4,22-stigmastadien-3-one (VIII), 3-oxo-4,22-stigmastadien-26-oic acid methyl ester (IX), and 3-oxo-1,4,22-stigmastatrien-26-oic acid methyl ester (X) with that strain. In both beta-sitosterol and stigmasterol, 26-oxidation generates the R-configuration on C-25.

Enantiomeric recognition of α-(1-naphthyl)ethylammonium perchlorate by enantiomerically pure dimethylphenazino-18-crown-6 ligand in solid and gas phases

Abstract X-Ray crystallographic studies of enantiomerically pure dimethylphenazino-18-crown-6 ligand (R,R)-1 and its complexes with the enantiomers of α-(1-naphthyl)ethylammonium perchlorate NapEt were carried out. These studies clearly show that the heterochiral complex (R,R)-1–(S)-NapEt is more stable than the homochiral one (R,R)-1–(R)-NapEt. It was pointed out that besides the hydrogen bonding, mainly the π–π interaction between the aromatic systems of the host and guest, and the difference in steric repulsions were responsible for enantioselectivity. Molecular mechanical calculations using the LMOD/MINTA method also predicted the heterochiral complex to be more stable than the homochiral one in the gas phase.

Structure determination and refinement of the Al3+ complex of the D254,256E mutant of Arthrobacter d-xylose isomerase at 2.40 Å resolution. Further evidence for inhibitor-induced metal ion movement

The structure of the D254.256E double mutant of Arthrobacter xylose isomerase with Al3+ at both metal-binding sites was determined by the molecular replacement method at a conventional R-factor of 0.179. Binding of the two Al3+ does not alter the overall structure significantly. However, there are local rearrangements in the octahedral co-ordination sphere of the Al3+. The inhibitor molecule moves somewhat away from the active site. Furthermore, evidence was revealed for metal ion movement from site 2(1) to site 2(2) upon double mutation. Xylose isomerase requires two divalent metal cations for activation. The catalytic metal ion is translocated 1.8 A away from its initial position during the catalytic reaction. The fact that both activating and inactivating metals (including Al3+) were found exclusively at a single location in the double mutant was an indication that the consequently missing shuttle may account for the crippled catalytic efficiency.