Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

Disaccharide Nucleosides and Oligonucleotides on Their Basis. New Tools for the Study of Enzymes of Nucleic Acid Metabolism

The main structural features of an important group of natural compounds, disaccharide nucleosides, are reviewed. The synthesis and properties of modified oligonucleotides on their basis as well as the methods of introduction of reactive aldehyde groups are described. The last part is devoted to the application of these compounds for studies of enzymes of nucleic acid metabolism.     

Carbon source utilization profiles as a method to identify sources of faecal pollution in water

AIMS: Carbon source utilization profiles as a phenotypic fingerprinting methodology for determining sources of faecal pollution in water were evaluated. METHODS AND RESULTS: Three hundred and sixty-five Enterococcus isolates were collected from known faecal sources in four different geographical regions and were identified to species with the commercial Biolog system. Discriminant analysis (DA) was used to identify the substrate-containing wells that best classified the 365 isolates by source. By using 30 of the 95 wells for the analysis, the average rate of correct classification (ARCC) by source was 92.7% for a human vs non-human two-way classification when isolates from all regions were combined into one library. Corresponding ARCCs for other classification schemes were 81.9% for a four-way classification of human vs livestock vs wildlife vs domestic pets, and 85.7% for a three-way classification without human isolates. When three individual libraries were made based on classification of sources within Enterococcus species, the ARCC was 95.3% for the Ent. faecalis library, 95.8% for the Ent. gallinarum library and 94.7% for the Ent. mundtii library. Thirty Enterococcus isolates (unknown sources) were obtained from each of three stream sites where a specific source of pollution was apparent; 90.0% of the isolates from a human-suspected source were classified as human, 86.6% were classified as livestock from a livestock-suspected site, and 93.3% were classified as wildlife from a wildlife-suspected site. CONCLUSIONS: Phenotypic fingerprinting with carbon source utilization profiles provided levels of correct classification by sources from an Enterococcus library that were in the upper range of those reported in the literature. ARCCs for three Enterococcus species-specific libraries were very high and may be the best approach for further developing this concept and methodology. SIGNIFICANCE ANC IMPACT OF THE STUDY: The results, based on a modest Enterococcus library and a preliminary field validation test, demonstrated the potential for carbon source utilization profiles to be employed as a phenotypic method for determining sources of faecal pollution in water.     

The value of a tree species (Caryocar brasiliense) for a stingless bee Melipona quadrifasciata quadrifasciata

The expansion of crop lands and increased logging for charcoal production in the Brazilian savannahs (cerrados) has reduced richness and abundance of Meliponini bees. This may be a consequence of limitation in the availability of potential nesting substrate. The role of a cerrado-tree (Caryocar brasiliense) in providing nesting substrate for Melipona quadrifasciata quadrifasciata was evaluated. Tree (p= 0.006) and branch (p= 0.001) diameters, number of suitable branches (n= 513), height of the trees and availability of trees suitable for bee nesting were all important to the conservation of M. quadrifasciata. However, the high availability of nesting substrate did not seem to limit nest density nor cause the clumped pattern of nest distributions found for the study site. Nests (n= 48) were found mainly in individuals of C. brasiliense (n= 46) suggesting an active tree selection. In addition, nests located on the highest branches (mean = 4.6 m, sd = 1 m, n= 46) had lower probability of being extirpated by human honey collectors.     

On the specificity of lipid hydroperoxide fragmentation by fatty acid hydroperoxide lyase from Arabidopsis thaliana

Fatty acid hydroperoxide lyase (HPL) is a membrane associated P450 enzyme that cleaves fatty acid hydroperoxides into aldehydes and omega-oxo fatty acids. One of the major products of this reaction is (3Z)-hexenal. It is a constituent of many fresh smelling fruit aromas. For its biotechnological production and because of the lack of structural data on the HPL enzyme family, we investigated the mechanistic reasons for the substrate specificity of HPL by using various structural analogues of HPL substrates. To approach this 13-HPL from Arabidopsis thaliana was cloned and expressed in E. coli utilising a His-Tag expression vector. The fusion protein was purified by affinity chromatography from the E. coli membrane fractions and its pH optimum was detected to be pH 7.2. Then, HPL activity against the respective (9S)- and (13S)-hydroperoxides derived either from linoleic, alpha-linolenic or gamma-linolenic acid, respectively, as well as that against the corresponding methyl esters was analysed. Highest enzyme activity was observed with the (13S)-hydroperoxide of alpha-linolenic acid (13alpha-HPOT) followed by that with its methyl ester. Most interestingly, when the hydroperoxy isomers of gamma-linolenic acid were tested as substrates, 9gamma-HPOT and not 13gamma-HPOT was found to be a better substrate of the enzyme. Taken together from these studies on the substrate specificity it is concluded that At13HPL may not recognise the absolute position of the hydroperoxy group within the substrate, but shows highest activities against substrates with a (1Z4S,5E,7Z)-4-hydroperoxy-1,5,7-triene motif. Thus, At13HPL may not only be used for the production of C6-derived volatiles, but depending on the substrate may be further used for the production of Cg-derived volatiles as well.     

Aerobic secondary utilization of a non-growth and inhibitory substrate 2,4,6-trichlorophenol by Sphingopyxis chilensis S37 and sphingopyxis-like strain S32

This paper reports 2,4,6-trichlorophenol (246TCP) degradation bySphingopyxis chilensis S37 and Sphingopyxis chilensis-like strain S32,which were unable to use 246TCP as the sole carbon and energy source. In R2A broth, the strainsdegraded 246TCP up to 0.5 mM. Results with mixtures of different 246TCP and glucose concentrations in mineral salt media demonstrated dependence on glucose to allow bacterial growth and degradation of 246TCP. Strain S32 degraded halophenol up to 0.2 mM when 5.33 mM glucose was simultaneously added, while strain S37 degraded the compound up to 0.1 mM when 1.33 mM glucose was added. These 246TCP concentrations were lethal for inocula in absence of glucose. Stoichiometricreleases of chloride and analysis by HPLC, GC-ECD and GC-MS indicated 246TCP mineralisation by both strains. To our knowledge, this is the first report of bacteriaable to mineralize a chlorophenol as a non-growth and inhibitory substrate. The concept of secondary utilization instead of cometabolism is proposed for this activity.     

Conformational studies on the MUC1 tandem repeat glycopeptides: implication for the enzymatic O-glycosylation of the mucin protein core

The tandem repeat of the MUC1 protein core is a major site of O-glycosylation that is catalyzed by several polypeptide GalNAc-transferases. To define structural features of the peptide substrates that contribute to acceptor substrate efficiency, solution structures of the 21-residue peptide AHGVTSAPDTRPAPGSTAPPA (AHG21) from the MUC1 protein core and four isoforms, glycosylated with alpha-N-acetylgalactosamine on corresponding Thr residues, AHG21 (T5), AHG21 (T10), AHG21 (T17), and AHG21 (T5,T17), were investigated by NMR spectroscopy and computational methods. NMR studies revealed that sugar attachment affected the conformational equilibrium of the peptide backbone near the glycosylated Thr residues. The clustering of the low-energy conformations for nonglycosylated and glycosylated counterparts within the VTSA, DTR, and GSTA fragments (including all sites of potential glycosylation catalyzed by GalNAc-T1, -T2, and -T4 transferases) showed that the glycosylated peptides display distinct structural propensities that may explain, in part, the differences in substrate specificities exhibited by these polypeptide GalNAc-transferases.     

Crystal structure of the catalytic domain of human bile salt activated lipase

Bile-salt activated lipase (BAL) is a pancreatic enzyme that digests a variety of lipids in the small intestine. A distinct property of BAL is its dependency on bile salts in hydrolyzing substrates of long acyl chains or bulky alcoholic motifs. A crystal structure of the catalytic domain of human BAL (residues 1-538) with two surface mutations (N186D and A298D), which were introduced in attempting to facilitate crystallization, has been determined at 2.3 A resolution. The crystal form belongs to space group P2(1)2(1)2(1) with one monomer per asymmetric unit, and the protein shows an alpha/beta hydrolase fold. In the absence of bound bile salt molecules, the protein possesses a preformed catalytic triad and a functional oxyanion hole. Several surface loops around the active site are mobile, including two loops potentially involved in substrate binding (residues 115-125 and 270-285).     

Polyelectrolyte-coated microcapsules and their potential applications to biotechnology

Polyelectrolyte-coated microcapsules can be prepared by adsorption of polyions onto microcapsule surfaces in aqueous solutions under appropriate pH and ionic conditions. The resulting polyelectrolyte-coated microcapsules provide a promising tool for studying pH-induced configurational changes in polyions adsorbed onto hydrophobic membranes (capsule walls). An interesting application of polyelectrolyte-coated microcapsules is the pH-sensitive on/off control of microencapsulated enzyme reactions through alterations in the substrate permeability of the capsule wall by pH-conditioned configurational changes in the adsorbed polyion layer. This paper presents an overview of pH-induced conformational changes of polyelectrolytes in solutions, preparation of polyelectrolyte-coated microcapsules with an immobilized enzyme, and on/off control of the respective enzyme reactions by pH adjustment. This revised version was published online in July 2006 with corrections to the Cover Date.     

Crystal structure of cis-biphenyl-2,3-dihydrodiol-2,3-dehydrogenase from a PCB degrader at 2.0 A resolution.

cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of polychlorinated biphenyls (PCBs). The crystal structure of the NAD+-enzyme complex was determined by molecular replacement and refined to an R-value of 17.9% at 2.0 A. As a member of the short-chain alcohol dehydrogenase/reductase (SDR) family, the overall protein fold and positioning of the catalytic triad in BphB are very similar to those observed in other SDR enzymes, although small differences occur in the cofactor binding site. Modeling studies indicate that the substrate is bound in a deep hydrophobic cleft close to the nicotinamide moiety of the NAD+ cofactor. These studies further suggest that Asn143 is a key determinant of substrate specificity. A two-step reaction mechanism is proposed for cis-dihydrodiol dehydrogenases.