Purification and properties of 4-hydroxyphenylacetic acid 3-hydroxylase from pseudomonas putida

4-Hydroxyphenylacetic acid 3-hydroxylase is a key enzyme in the pathway for the microbial degradation of phenylalanine, tyrosine and many aromatic amines. This enzyme was purified to homogeneity from Image by affinity chromatography. The protein had a molecular weight of 91,000 and was a dimer of identical subunits. It was a typical external flavoprotein monooxygenase and showed an absolute requirement of NADH for activity. The enzyme had a pH optimum of 7.5 and the Km values for 4-hydroxyphenylacetic acid and NADH were 2×10−4 M and 5.9×10−5 M respectively. It was strongly inhibited by heavy metal ions and thiol reagents, suggesting the possible involvement of -SH group(s) in enzyme reaction.

Structure of 1,2,4-oxadiazolidine-3,5-dione monohydrate

C2H2N203.H20, Mr= 120.07, monoclinic,P21/c, a= 5.011 (1), b= 11.796(2), c= 7.689 (2)A,fl= 95.22 (2) ° , V= 452.61 A 3, Z= 4, Dx= 1.76, D m = 1.75 gcm -3, /].(Cu Ks) = 1.5418 A, g = 14-0 cm -l,F(000) = 248, T = 293 K, crystal quality was poor and the final R =0.107, wR =0.090 for 881 observed reflections. The compound is derived from a novel form of the monopropellant oxalohydroxamic acid. The two exocyclic C-O bond lengths of 1.240 (3) and 1.228 (4)A indicate double bonds. The C-N bond lengths of 1.334 (4), 1.390 (4) and 1.359 (4) A are characteristic of the amide bond. The N atom covalently bonded to the two carbonyl C atoms acts as a proton donor in an intermolecular hydrogen bond to the ring O atom: N1...O3i = 2.854 ]k (i =x-- 1,y, z), H...O = 2.15 A, N-H...O = 159 °.

Structure of L-Arginyl-L-aspartie Acid Dihydrate

C~0H~gN5Os.2H20, Mr=325.32, monoclinic,P2~, a = 12.029 (2), b=4.904 (2), c=13.215 (2) A, fl= 107.68 (2) ° , F= 743 (1) A 3, Z= 2,D m = 1-45, D x = 1.45 Mg m -3, Cu Ka, 2 = 1.54184 A,fl= 1.01mm -1, F(000)=348, T=293K. The final R value for 1277 observed reflections 110 >_ 3tr(Io)l is 0.031. The dipeptide exists as a zwitterion. The arginyl side-chain conformation is similar to that found in arginyl-glutamic acid [Pandit, Seshadri & Viswamitra (1983). Acta Cryst. C39, 1669-16721. The guanidyl group forms a pair of hydrogen bonds with oxygen atoms of the backbone carboxyl group. The crystal structure is also stabilized by -bonding interactions involving both water molecules.

Synthesis, Structure and Optical Studies of a Family of Three-Dimensional Rare-Earth Aminoisophthalates M(mu(2)-OH)(C8H5NO4)] (M = Y3+, La3+, Pr3+, Nd3+, Sm3+, Eu3+, Gd3+, Dy3+, and Er3+)

A hydrothermal reaction of the acetate salts of the rare-earths, 5-aminoisophthalic acid (H(2)AIP), and NaOH at 150 degrees C for 3 days gave rise to a new family of three-dimensional rare-earth aminoisophthalates, M(mu(2)-OH)(C8H5NO4)] M = Y3+ (I), La3+ (II), Pr3+ (III), Nd3+ (IV), Sm3+ (V), Eu3+ (VI), Gd3+ (VII), Dy3+ (VIII), and Er3+ (IX)]. The structures contain M-O(H)-M chains connected by AIP anions. The AIP ions are connected to five metal centers and each metal center is connected with five AIP anions giving rise to a unique (5,5) net. To the best of our knowledge, this is the first observation of a (5,5) net in metal-organic frameworks that involve rare-earth elements. The doping of Eu3+/(3+) ions in place of Y3+/ La3+ in the parent structures gave rise to characteristic metal-centered emission (red = Eu3+, green = Tb3+). Life-time studies indicated that the excited emission states in the case of Eu3+ (4 mol-% doped) are in the range 0.287-0.490 ms and for Tb3+ (4 mol-% doped) are in the range of 1.265-1.702 ms. The Nd3+-containing compound exhibits up-conversion behavior based on two-photon absorption when excited using lambda = 580 nm.

2,1,3-Bentsoksadiatsolirakenteiset molekyylit antibakteerisina ja antiparasiittisina yhdisteinä

Aikaisemman tutkimuksen perusteella tiedettiin tiettyjen 2,1,3-bentsoksadiatsolirakenteisten molekyylien olevan aktiivisia Chlamydia pneumoniae –bakteeria vastaan. Tutkimusta lähdettiin jatkamaan ja 2,1,3-bentsoksadiatsolimolekyylien rakenne-aktiivisuusuhteista haluttiin saada lisätietoa. Tarkoituksena oli kehittää 2,1,3-bentsoksadiatsolimolekyyleille ja sen avulla muodostaa molekyylikirjasto. Syntetisoidut molekyylit haluttiin testata sekä Chlamydia pneumoniae -bakteeria että Leishmania donovani –parasiittia vastaan. Chlamydia pneumoniae –bakteeri aiheuttaa akuutteja ylä- ja alahengitystieinfektiota, kuten keuhkoputkentulehdusta. Akuutissa tulehduksessa oireet vaihtelevat huomattavasti. Chlamydia pneumoniae –bakteerilla on myös taipumus aiheuttaa kroonisia tulehduksia. Nämä ovat useissa tutkimuksissa yhdistetty kansantaloudellisesti merkittäviin sairauksiin, kuten ateroskleroosiin ja astmaan. Leishmanioosi on toiseksi yleisin loissairaus ihmisellä malarian jälkeen. Leishmania donovani –parasiitti voi aiheuttaa tappavaa viskeraalista leishmanioosia. Vuodessa leishmanioosiin kuolee yli 50 000 ihmistä. Viime vuosina leishmanioosin lääkehoidossa on esiintynyt monenlaisia ongelmia. Osat lääkkeistä ovat menettäneet tehonsa ja osalla esiintyy vakavia haittavaikutuksia. 2,1,3-Bentsoksadiatsolirakenteisille yhdisteille saatiin kehitettyä toimiva synteesireitti. Lähtöaineena käytettiin 4-amino-2-nitrobentsoehappoa, josta saatiin hapettavalla renkaansulkeutumisreaktiolla 2,1,3-bentsoksadiatsoli-5-karboksyylihappoa. Karboksyylihaposta syntetisoitiin amidi-välituotteen kautta 2,1,3-bentsoksadiatsoli-5-karbonitriiliä. Hydroksyyliamiini hydrokloridin avulla 2,1,3-bentsoksadiatsoli-5-karbonitriilistä muodostettiin vastaavaa karboksimidamidia, joka oli synteesireitin yhteinen välituote kaikille molekyyleille. Viimeisessä vaiheessa N´-hydroksidi-2,1,3-bentsoksadiatsoli-5-karboksimidamidin annettiin reagoida joko fenyyli-isosyanaatin tai fenyyli-isotiosyanaatin kanssa, jolloin saatiin lopputuotetta. Synteesireitin kehittäminen osoittautui haastavaksi ja loppujen lopuksi saatiin ainoastaan kolme lopputuotetta syntetisoitua. Yksi lopputuotteista testattiin C. pneumoniae –bakteeria vastaan Åbo akademissa Turussa. Testattavaa yhdiste ei sisältänyt 2,1,3-bentsoksadiatsoliarengasta ja bioaktiivisuuskokeen tulos oli odotusten mukainen. Yhdiste ei ollut aktiivinen C. pneumoniae –bakteeria vastaan alhaisilla konsentraatioilla ja tuloksesta voitiin todeta 2,1,3-bentsoksadiatsolirengaan olevan tärkeä aktiivisuuden kannalta. Kaksi lopputuotetta saatiin testaukseen Leishamania donovani –parasiittia vastaan Israeliin. Ainoastaan toinen molekyyleistä sisälsi 2,1,3-bentsoksadiatsolirakenteen. Bioaktiivisuuskokeiden tulokset olivat erittäin rohkaisevia. Yhdisteet olivat aktiivisia parasiittia vastaan jo alhaisilla konsentraatioilla. Kuitenkin 2,1,3-bentsoksadiatsolirakenteinen molekyyli oli aktiivisempi, joten tämäkin aktiivisuuskokeen perusteella huomattiin rengasrakenteen olevan tärkeä aktiivisuuden kannalta.

Stereochemically constrained peptides. Theoretical and experimental studies on the conformations of peptides containing 1-aminocyclohexane carboxylic acid

Conformational energy calculations on the model system N-acetyl- 1 -aminocyclohexanecarboxylic acid N'methylamide (Ac-Acc6-NHMe), using an average geometry derived from 13 crystallographic observations, establish that the Acc6 residue is constrained to adopt conformations in the helical regions of In contrast, the a,a-dialkylated residue with linear hydrocarbon side chains, a,a-di-n-propylglycine favors fully extended backbone structures (6 1= $ = 180'). The crystal structures of two model peptides, Boc-(Acc6),-OMe (type 111 @-turn at -Acc6(1)-Acc6(2)-) and Boc-Pro-Acc6-Ala-OMe (type I1 P-turn at -Pro-Acc6-), establish that Acc6 residues can occupy either position of type 111 P-turns and the i + 2 position of type I1 @-turns. The stereochemical rigidity of these peptides is demonstrated in solution by NMR studies, which establish the presence of one intramolecular hydrogen bond in each peptide in CDCI, and (CDJ2S0. Nuclear Overhauser effects permit characterization of the @-turn conformations in solution and establish their similarity to the solid-state structures. The implications for the use of Acc6 residues in conformational design are considered.

Crystallographic characterization of the conformation of the 1-aminocyclohexane-1-carboxylic acid residue in simple derivatives and peptides

The crystal structures of 1-aminocyclohexane-1-carboxylic acid (H-Acc6-OH) and six derivatives (including dipeptides) have been determined. The derivatives are Boc-Acc6-OH, Boc-(Acc6)2-OH, Boc-L-Met-Acc6-OMe, ClCH2CO-Acc6-OH, p-BrC6H4CO-Acc6-OH oxazolone, and the symmetrical anhydride from Z-Acc6-OH, [(Z-Acc6)2O]. The cyclohexane rings in all the structures adopt an almost perfect chair conformation. The amino group occupies the axial position in six structures; the free amino acid is the only example where the carbonyl group occupies an axial position. The values determined for the torsion angles about the N–Cα(φ) and Cα–CO (ψ) bonds correspond to folded, potentially helical conformations for the Acc6 residue.

Empirical torsional potential functions from protein structure data. Phi- and psi-potentials for non-glycyl amino acid residues.

The torsional potential functions Vt(phi) and Vt(psi) around single bonds N--C alpha and C alpha--C, which can be used in conformational studies of oligopeptides, polypeptides and proteins, have been derived, using crystal structure data of 22 globular proteins, fitting the observed distribution in the (phi, psi)-plane with the value of Vtot(phi, psi), using the Boltzmann distribution. The averaged torsional potential functions, obtained from various amino acid residues in L-configuration, are Vt(phi) = 1.0 cos (phi + 60 degrees); Vt(psi) = 0.5 cos (psi + 60 degrees) - 1.0 cos (2 psi + 30 degrees) - 0.5 cos (3 psi + 30 degrees). The dipeptide energy maps Vtot(phi, psi) obtained using these functions, instead of the normally accepted torsional functions, were found to explain various observations, such as the absence of the left-handed alpha helix and the C7 conformation, and the relatively high density of points near the line psi = 0 degrees. These functions derived from observational data on protein structures, will, it is hoped, explain various previously unexplained facts in polypeptide conformation.

Affinity purification and characterization of 2,4-dichlorophenol hydroxylase from Pseudomonas cepacia

2,4-Dichlorophenol hydroxylase, a flavoprotein monooxygenase from Pseudomonas cepacia grown on 2,4-dichlorophenoxyacetic acid (2,4-D) as the sole source of carbon, was purified to homogeneity by a single-step affinity chromatography on 2,4-DCP-Sepharose CL-4B. The enzyme was eluted from the affinity matrix with the substrate 2,4-dichlorophenol. The enzyme has a molecular weight of 275,000 consisting of four identical subunits of molecular weight 69,000 and requires exogenous addition of FAD for its complete catalytic activity. The enzyme required an external electron donor NADPH for hydroxylation of 2,4-dichlorophenol to 3,5-dicholorocatechol. NADPH was preferred over NADH. The enzyme had Km value of 14 μImage for 2,4-dichlorophenol, and 100 μImage for NADPH. The enzyme activity was significantly inhibited by heavy metal ions like Hg2+ and Zn2+ and showed marked inhibition with thiol reagents. Trichlorophenols inhibited the enzyme competitively. The hydroxylase activity decreased as a function of increasing concentrations of Cibacron blue and Procion red dyes. The apoenzyme prepared showed complete loss of FAD when monitored spectrophotometrically and had no enzymatic activity. The inactive apoenzyme was reconstituted with exogenous FAD which completely restored the enzyme activity.

Crystal structure of hexanediamine-glutamic acid complex

Polyamines are some of the most important and ubiquitous small molecules that modulate several functions of plant, animal and bacterial cells. Despite the simplicity of their chemical structure, their specific interactions with other biomolecules cannot be explained solely on the basis of their electrostatic properties. To evolve a structural understanding on the specificity of these interactions it is necessary to determine the structure of complexes of polyamines with other, representative biomolecules. This paper reports the structure of the 1:2 complex of hexanediamine and L-glutamic acid. The complex crystallizes in the monoclonic space group P2(1) with a = 5.171(1) angstrom, b = 22.044(2) angstrom, c = 10.181(2) angstrom and beta = 104.51(1)-degrees. The structure was refined to an R factor of 6.6%. The structures of these complexes not only suggest the importance of hydrogen-bonding interactions of polyamines but also provide some insight into other complementary interactions probably important for the specificity of biomolecular interactions.

A nonhelical, multiple β-turn conformation in a glycine-rich heptapeptide fragment of trichogin A IV containing a single central α-aminoisobutyric acid residue

The conformational properties of the protected seven-residue C-terminal fragment the lipopeptaibol antibiotic Trichogin A IV (Boc-Gly-Gly-Leu-Aib-Gly-Ile-Leu-OMe) has been examined in CDCl3 and (CD3)2SO by 1H-nmr. Evidence for a multiple β-turn conformation [type I′ at Gly(1)-Gly(2), type II at Leu(3)-Aib(4), and a type I′ at Aib(4)-Gly(5)] suggests that Leu(3) has preferred an extended or semiextended conformation over a helical conformation in CDCl3. This structure is thus in contrast to earlier observations of seven-residue peptides containing a single central Aib preferring helical conformations in both solution and crystalline slates. A structural transition to a frayed right-handed helix is absented in (CD3)2SO. These results suggest that nonhelical conformations may be important in Gly-rich peptides containing Aib. Further, the presence of amino acids with contradictory influences on backbone conformational freedom can lead to well-defined conformational transitions even in small peptides

Synthetic studies in aromatic hemiterpenes of natural origin, Part 5: Synthesis of 6-acetyl-2,2-dimethyl-8-methoxychromene via benzylic oxidation route

The synthesis of 6-acetyl-2,2-dimethyl-8-methoxychromene (lc), a naturally occurring isomer of encecalin (la)h~s been described startilag from 2,2,6- trimethyl-8-methoxyclaromene (2e) which was obtained from creosol (4) in two steps involving condensation of the phenol with malic acid to the coumarin (3), followed by Grignard reaction with CHaMgI. The transformation of (2e) to the natural product (lc) was effeeted by oxidative dehydrogenation by DDQ of the 6-meth~r function to the formyl group (2f), Grignard reaction to the carbinol (2g) and finally its oxidation to the acetyl moiety (lc), the sequence of the essential steps schematically summarised as : Ar-CHs --* Ar-CHO --* Ar-CH (OH) CHs --* Ar---COCHs.

Effect of amino acid substitutions at the subunit interface on the stabiity and aggregation properties of a dimeric protein: role of Arg 178 and Arg 218 at the dimer interface of thymidylate synthase

The significance of two interface arginine residues on the structural integrity of an obligatory dimeric enzyme thymidylate synthase (TS) from Lactobacillus casei was investigated by thermal and chemical denaturation. While the R178F mutant showed apparent stability to thermal denaturation by its decreased tendency to aggregate, the Tm of the R218K mutant was lowered by 5 degrees C. Equilibrium denaturation studies in guanidinium chloride (GdmCl) and urea indicate that in both the mutants, replacement of Arg residues results in more labile quaternary and tertiary interactions. Circular dichroism studies in aqueous buffer suggest that the protein interior in R218K may be less well-packed as compared to the wild type protein. The results emphasize that quaternary interactions may influence the stability of the tertiary fold of TS. The amino acid replacements also lead to notable alteration in the ability of the unfolding intermediate of TS to aggregate. The aggregated state of partially unfolded intermediate in the R178F mutant is stable over a narrower range of denaturant concentrations. In contrast, there is an exaggerated tendency on the part of R218K to aggregate in intermediate concentrations of the denaturant. The 3 A crystal structure of the R178F mutant reveals no major structural change as a consequence of amino acid substitution. The results may be rationalized in terms of mutational effects on both the folded and unfolded state of the protein. Site specific amino acid substitutions are useful in identifying specific regions of TS involved in association of non-native protein structures.