Neopinone isomerase is involved in codeine and morphine biosynthesis in opium poppy.

The isomerization of neopinone to codeinone is a critical step in the biosynthesis of opiate alkaloids in opium poppy. Previously assumed to be spontaneous, the process is in fact catalyzed enzymatically by neopinone isomerase (NISO). Without NISO the primary metabolic products in the plant, in engineered microbes and in vitro are neopine and neomorphine, which are structural isomers of codeine and morphine, respectively. Inclusion of NISO in yeast strains engineered to convert thebaine to natural or semisynthetic opiates dramatically enhances formation of the desired products at the expense of neopine and neomorphine accumulation. Along with thebaine synthase, NISO is the second member of the pathogenesis-related 10 (PR10) protein family recently implicated in the enzymatic catalysis of a presumed spontaneous conversion in morphine biosynthesis.

On the substrate specificity of the rice strigolactone biosynthesis enzyme DWARF27.

MAIN CONCLUSION: The ß-carotene isomerase OsDWARF27 is stereo- and double bond-specific. It converts bicyclic carotenoids with at least one unsubstituted ß-ionone ring. OsDWARF27 may contribute to the formation of α-carotene-based strigolactone-like compounds. Strigolactones (SLs) are synthesized from all-trans-ß-carotene via a pathway involving the ß-carotene isomerase DWARF27, the carotenoid cleavage dioxygenases 7 and 8 (CCD7, CCD8), and cytochrome P450 enzymes from the 711 clade (MAX1 in Arabidopsis). The rice enzyme DWARF27 was shown to catalyze the reversible isomerization of all-trans- into 9-cis-ß-carotene in vitro. ß-carotene occurs in different cis-isomeric forms, and plants accumulate other carotenoids, which may be substrates of DWARF27. Here, we investigated the stereo and substrate specificity of the rice enzyme DWARF27 in carotenoid-accumulating E. coli strains and in in vitro assays performed with heterologously expressed and purified enzyme. Our results suggest that OsDWARF27 is strictly double bond-specific, solely targeting the C9-C10 double bond. OsDWARF27 did not introduce a 9-cis-double bond in 13-cis- or 15-cis-ß-carotene. Substrates isomerized by OsDWARF27 are bicyclic carotenoids, including ß-, α-carotene and ß,ß-cryptoxanthin, that contain at least one unsubstituted ß-ionone ring. Accordingly, OsDWARF27 did not produce the abscisic acid precursors 9-cis-violaxanthin or -neoxanthin from the corresponding all-trans-isomers, excluding a direct role in the formation of this carotenoid derived hormone. The conversion of all-trans-α-carotene yielded two different isomers, including 9'-cis-α-carotene that might be the precursor of strigolactones with an ε-ionone ring, such as the recently identified heliolactone.

Molybdopterin biosynthesis-Mechanistic studies on a novel MoaA catalyzed insertion of a purine carbon into the ribose of GTP.

The first step in the biosynthesis of the molybdopterin cofactor involves an unprecedented insertion of the purine C8 carbon between the C2' and C3' carbons of the ribose moiety of GTP. Here we review mechanistic studies on this remarkable transformation. This article is part of a Special Issue entitled: Cofactor-dependent proteins: evolution, chemical diversity and bio-applications.

A universal RNA polymerase II CTD cycle is orchestrated by complex interplays between kinase, phosphatase, and isomerase enzymes along genes.

Transcription by RNA polymerase II (RNAPII) is coupled to mRNA processing and chromatin modifications via the C-terminal domain (CTD) of its largest subunit, consisting of multiple repeats of the heptapeptide YSPTSPS. Pioneering studies showed that CTD serines are differentially phosphorylated along genes in a prescribed pattern during the transcription cycle. Genome-wide analyses challenged this idea, suggesting that this cycle is not uniform among different genes. Moreover, the respective role of enzymes responsible for CTD modifications remains controversial. Here, we systematically profiled the location of the RNAPII phosphoisoforms in wild-type cells and mutants for most CTD modifying enzymes. Together with results of in vitro assays, these data reveal a complex interplay between the modifying enzymes, and provide evidence that the CTD cycle is uniform across genes. We also identify Ssu72 as the Ser7 phosphatase and show that proline isomerization is a key regulator of CTD dephosphorylation at the end of genes.

Competitive enzymatic interactions determine the relative amounts of prostaglandins E2 and D2.

Prostaglandins (PGs) are a family of cellular messengers exerting diverse homeostatic and pathophysiologic effects. Recently, several studies reported significant increases of PGI(2) and PGF(2α) after the inhibition of microsomal PGE synthase-1 (mPGES-1) expression, which indicated that PGH(2) metabolism might be redistributed when the PGE(2) pathway is blocked. To address the determinants that govern the relative amounts of PGs, we developed an in vitro cell-free method, based on liquid chromatography-tandem mass spectrometry, to measure the exact amounts of these PGs formed in response to the addition of recombinant isomerases and their selective inhibitors. Our in vitro cell-free assay results were confirmed in cells using bone marrow-derived macrophage. Initially, we determined the in vitro stability of PGH(2) and noted that there was spontaneous nonenzymatic conversion to PGD(2) and PGE(2). mPGES-1 markedly increased the conversion to PGE(2) and decreased conversion to PGD(2). Reciprocally, the addition of hematopoietic or lipocalin PGD synthase resulted in a relative increase of PGD(2) and decrease of PGE(2). A detailed titration study showed that the ratio of PGE(2)/PGD(2) was closely correlated with the ratio of PGE synthase/PGD synthase. Our redistribution results also provide the foundation for understanding how PGH(2) metabolism is redistributed by the presence of distal isomerases or by blocking the major metabolic outlet, which could determine the relative benefits and risks resulting from interdiction in nonrated-limiting components of PG synthesis pathways.

Kinetic, crystallographic, and mechanistic characterization of TomN: elucidation of a function for a 4-oxalocrotonate tautomerase homologue in the tomaymycin biosynthetic pathway.

The biosynthesis of the C ring of the antitumor antibiotic agent, tomaymycin, is proposed to proceed through five enzyme-catalyzed steps from l-tyrosine. The genes encoding these enzymes have recently been cloned and their functions tentatively assigned, but there is limited biochemical evidence supporting the assignments of the last three steps. One enzyme, TomN, shows 58% pairwise sequence similarity with 4-oxalocrotonate tautomerase (4-OT), an enzyme found in a catabolic pathway for aromatic hydrocarbons. The TomN sequence includes three amino acids (Pro-1, Arg-11, and Arg-39) that have been identified as critical catalytic residues in 4-OT. However, the proposed substrate for TomN is very different from that processed by 4-OT. To establish the function and mechanism of TomN and its relationship with 4-OT, we conducted kinetic, mutagenic, and structural studies. The kinetic parameters for TomN, and four alanine mutants, P1A, R11A, R39A, and R61A, were determined using 2-hydroxymuconate, the substrate for 4-OT. The TomN-catalyzed reaction using this substrate compares favorably to that of 4-OT. In addition, the kinetic parameters for the P1A, R11A, and R39A mutants of TomN parallel the trends observed for the corresponding 4-OT mutants, implicating an analogous mechanism. A high-resolution crystal structure (1.4 Å) of TomN shows that the overall structure and the active site region are highly similar to those of 4-OT with a root-mean-square deviation of 0.81 Å. Moreover, key active site residues are positionally conserved. The combined results suggest that the tentative assignment for TomN and the proposed sequence of events in the biosynthetic pathway leading to the formation of the C ring of tomaymycin might not be correct. An alternative pathway that awaits biochemical confirmation is proposed.

Calnexin and ERp57 facilitate the assembly of the neonatal Fc receptor for IgG with beta 2-microglobulin in the endoplasmic reticulum.

The neonatal FcR (FcRn) consists of an MHC class I-like H chain in noncovalent association with beta(2)-microglobulin (beta(2)m). The proper folding of FcRn in the endoplasmic reticulum is essential for FcRn function. Using a low stringency immunoprecipitation of human FcRn, we observed the coprecipitation of an 88-kDa band. Mass spectrometry analysis revealed that this band was identical with calnexin (CNX). This association was verified by Western blotting the CNX or FcRn immunoprecipitates with either an anti-FcRn or anti-CNX Ab. In the beta(2)m-null FO-1 cell transfected with FcRn H chain alone or both FcRn H chain and beta(2)m, CNX bound to the FcRn H chain before the FcRn H chain association with beta(2)m. However, calreticulin only bound to the FcRn H chain-beta(2)m complex. Furthermore, the thiol oxidoreductase ERp57 was detected in FcRn-CNX complexes, suggesting its role in disulfide bond formation of the FcRn H chain. Removal of the N-linked glycosylation site from the FcRn H chain resulted in a decreased association of the FcRn H chain for beta(2)m. However, the absence of CNX did not significantly affect FcRn assembly as defined by the ability of FcRn to bind IgG and exit to the cell surface. This suggests that other chaperones compensate for the function of CNX in FcRn assembly. In addition, we found that tapasin and TAP were not involved in FcRn assembly, as shown by coimmunoprecipitation in THP-1 cells and IgG-binding assays in 721.220 (tapasin-deficient) and 721.174 (TAP-deficient) cells transfected with FcRn. These findings show the importance of chaperones in FcRn assembly.

The 1,25D3-MARRS protein: contribution to steroid stimulated calcium uptake in chicks and rats.

There are currently two main candidates for the membrane receptor for 1,25(OH)2D3: the 1,25D3-MARRS protein/ERp57; and the classical VDR. The 1,25D3-MARRS protein is essential for hormone-stimulated phosphate and calcium uptake in chick intestinal cells, whereas the VDR is not. The 1,25D3-MARRS protein also shows a high degree of correlation with growth periods in which bone is rapidly formed, whereas the VDR does not. However, in rat enterocytes, both the 1,25D3-MARRS protein and the VDR play a role in the rapid, steroid-mediated uptake of phosphate or calcium. Therefore, the theory that alternate binding sites on the VDR for various analogs account for all membrane-initiated phenomena, is incorrect.

Identification and characterization of 1,25D3-membrane-associated rapid response, steroid (1,25D3-MARRS)-binding protein in rat IEC-6 cells.

We report the presence of a mammalian equivalent of the avian Membrane-Associated Rapid Response, Steroid (1,25D3-MARRS)-binding protein specific for 1,25(OH)2D3 in a rat small intestinal cell line, IEC-6, that demonstrates rapid responses to the steroid hormone. Identification of transcript and protein was achieved using RT-PCR with several specific primer sets, Western blot analysis with two separate antibodies recognizing distinct regions of the protein, ribozyme knockdown and immunohistochemistry. Promoter analysis of the 1000-bp upstream region of the 1,25D3-MARRS gene in several species revealed the presence of a conserved smad-3 element in the 5' proximal promoter region, but no classical vitamin D response element (VDRE). Treatment of IEC-6 cells with transforming growth factor beta1 (TGFbeta1) increased steady-state levels of 1,25D3-MARRS (mRNA and protein) approximately two-fold over a 24-h period. In contrast, treatment with 1,25(OH)2D3 failed to significantly change 1,25D3-MARRS protein or mRNA levels. Localization studies showed rapid nuclear translocation of a pool of 1,25D3-MARRS protein after 1,25(OH)2D3 treatment, suggesting that the protein is subject to membrane-initiated signal pathway activation. Together these data point to complex interactions between the two important 1,25(OH)2D3 sensitive response systems in intestinal cells, 1,25D3-MARRS protein and the well-studied nVDR, that together work to fine tune intestinal Ca2+ absorption in a variety of avian and mammalian species.

Developmental changes of bile acid composition and conjugation in L- and D-bifunctional protein single and double knockout mice.

Peroxisomal beta-oxidation is an essential step in bile acid synthesis, since it is required for shortening of C27-bile acid intermediates to produce mature C24-bile acids. D-Bifunctional protein (DBP) is responsible for the second and third step of this beta-oxidation process. However, both patients and mice with a DBP deficiency still produce C24-bile acids, although C27-intermediates accumulate. An alternative pathway for bile acid biosynthesis involving the peroxisomal L-bifunctional protein (LBP) has been proposed. We investigated the role of LBP and DBP in bile acid synthesis by analyzing bile acids in bile, liver, and plasma from LBP, DBP, and LBP:DBP double knock-out mice. Bile acid biosynthesis, estimated by the ratio of C27/C24-bile acids, was more severely affected in double knock-out mice as compared with DBP-/- mice but was normal in LBP-/- mice. Unexpectedly, trihydroxycholestanoyl-CoA oxidase was inactive in double knock-out mice due to a peroxisomal import defect, preventing us from drawing any firm conclusion about the potential role of LBP in an alternative bile acid biosynthesis pathway. Interestingly, the immature C27-bile acids in DBP and double knock-out mice remained unconjugated in juvenile mice, whereas they occurred as taurine conjugates after weaning, probably contributing to the minimal weight gain of the mice during the lactation period. This correlated with a marked induction of bile acyl-CoA:amino acid N-acyltransferase expression and enzyme activity between postnatal days 10 and 21, whereas the bile acyl-CoA synthetases increased gradually with age. The nuclear receptors hepatocyte nuclear factor-4alpha, farnesoid X receptor, and peroxisome proliferator receptor alpha did not appear to be involved in the up-regulation of the transferase.

Glutathione directly reduces an oxidoreductase in the endoplasmic reticulum of mammalian cells.

The formation of disulfide bonds is an essential step in the folding of many glycoproteins and secretory proteins. Non-native disulfide bonds are often formed between incorrect cysteine residues, and thus the cell has dedicated a family of oxidoreductases that are thought to isomerize non-native bonds. For an oxidoreductase to be capable of performing isomerization or reduction reactions, it must be maintained in a reduced state. Here we show that most of the oxidoreductases are predominantly reduced in vivo. Following oxidative stress the oxidoreductases are quickly reduced, demonstrating that a robust reductive pathway is in place in mammalian cells. Using ERp57 as a model we show that the reductive pathway is cytosol-dependent and that the component responsible for the reduction of the oxidoreductases is the low molecular mass thiol glutathione. In addition, ERp57 is not reduced following oxidative stress when inhibitors of glutathione synthesis or glutathione reduction are added to cells. Glutathione directly reduces ERp57 at physiological concentrations in vitro, and biotinylated glutathione forms a mixed disulfide with ERp57 in microsomes. Our results demonstrate that glutathione plays a direct role in the isomerization of disulfide bonds by maintaining the mammalian oxidoreductases in a reduced state.

Tapasin enhances MHC class I peptide presentation according to peptide half-life.

Understanding how peptides are selected for presentation by MHC class I is crucial to vaccination strategies based on cytotoxic T lymphocyte priming. We have studied this selection of the MHC class I peptide repertoire in terms of the presentation of a series of individual peptides with a wide range of binding to MHC class I. This series was expressed as minigenes, and the presentation of each peptide variant was determined with the same MHC class I peptide-specific antibody. In wild-type cells, the hierarchy of presentation followed peptide half-life. This hierarchy broke down in cells lacking tapasin but not in cells lacking calreticulin or in cells lacking transporter associated with antigen processing-associated ERp57. We demonstrate a key role for tapasin in shaping the MHC class I peptide repertoire, as enhancement of presentation in the presence of tapasin correlated with peptide half-life.

The fabM gene product of Streptococcus mutans is responsible for the synthesis of monounsaturated fatty acids and is necessary for survival at low pH.

Previously, it has been demonstrated that the membrane fatty acid composition of Streptococcus mutans is affected by growth pH (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004; R. G. Quivey, Jr., R. Faustoferri, K. Monahan, and R. Marquis, FEMS Microbiol. Lett. 189:89-92, 2000). Specifically, the proportion of monounsaturated fatty acids increases when the organism is grown in acidic environments; if the shift to increased monounsaturated fatty acids is blocked by the addition of a fatty acid biosynthesis inhibitor, the organism is rendered more acid sensitive (E. M. Fozo and R. G. Quivey, Jr., Appl. Environ. Microbiol. 70:929-936, 2004). Recently, work with Streptococcus pneumoniae has identified a novel enzyme, FabM, responsible for the production of monounsaturated fatty acids (H. Marrakchi, K. H. Choi, and C. O. Rock, J. Biol. Chem. 277:44809-44816, 2002). Using the published S. pneumoniae sequence, a putative FabM was identified in the S. mutans strain UA159. We generated a fabM strain that does not produce unsaturated fatty acids as determined by gas chromatography of fatty acid methyl esters. The mutant strain was extremely sensitive to low pH in comparison to the wild type; however, the acid-sensitive phenotype was relieved by growth in the presence of long-chain monounsaturated fatty acids or through genetic complementation. The strain exhibited reduced glycolytic capability and altered glucose-PTS activity. In addition, the altered membrane composition was more impermeable to protons and did not maintain a normal DeltapH. The results suggest that altered membrane composition can significantly affect the acid survival capabilities, as well as several enzymatic activities, of S. mutans.

Separate roles and different routing of calnexin and ERp57 in endoplasmic reticulum quality control revealed by interactions with asialoglycoprotein receptor chains.

The thiol oxidoreductase endoplasmic reticulum (ER)p57 interacts with newly synthesized glycoproteins through ternary complexes with the chaperones/lectins calnexin or calreticulin. On proteasomal inhibition calnexin and calreticulin concentrate in the pericentriolar endoplasmic reticulum-derived quality control compartment that we recently described. Surprisingly, ERp57 remained in an endoplasmic reticulum pattern. Using asialoglycoprotein receptor H2a and H2b as models, we determined in pulse-chase experiments that both glycoproteins initially bind to calnexin and ERp57. However, H2b, which will exit to the Golgi, dissociated from calnexin and remained bound for a longer period to ERp57, whereas the opposite was true for the endoplasmic reticulum-associated degradation substrate H2a that will go to the endoplasmic reticulum-derived quality control compartment. At 15 degrees C, ERp57 colocalized with H2b adjacent to an endoplasmic reticulum-Golgi intermediate compartment marker. Posttranslational inhibition of glucose excision prolonged association of H2a precursor to calnexin but not to ERp57. Preincubation with a low concentration (15 microg/ml) of the glucosidase inhibitor castanospermine prevented the association of H2a to ERp57 but not to calnexin. This low concentration of castanospermine accelerated the degradation of H2a, suggesting that ERp57 protects the glycoprotein from degradation and not calnexin. Our results suggest an early chaperone-mediated sorting event with calnexin being involved in the quality control retention of molecules bound for endoplasmic reticulum-associated degradation and ERp57 giving initial protection from degradation and later assisting the maturation of molecules that will exit to the Golgi.

Biosynthesis of deoxyaminosugars in antibiotic-producing bacteria.

Deoxyaminosugars comprise an important class of deoxysugars synthesized by a variety of different microorganisms; they can be structural components of lipopolysaccharides, extracellular polysaccharides, and secondary metabolites such as antibiotics. Genes involved in the biosynthesis of the deoxyaminosugars are often clustered and are located in the vicinity of other genes required for the synthesis of the final compound. Most of the gene clusters for aminosugar biosynthesis have common features, as they contain genes encoding dehydratases, isomerases, aminotransferases, methyltransferases, and glycosyltransferases. In the present mini-review, the proposed biosynthetic pathways for deoxyaminosugar components of both macrolide and non-macrolide antibiotics are highlighted. The possibilities for genetic manipulations of the deoxyaminosugar biosynthetic pathways aimed at production of novel secondary metabolites are discussed.

Role of GRP58 in mitomycin C-induced DNA cross-linking.

Mitomycin C (MMC) is an anticancer drug that requires reductive activation to exert its toxicity. MMC is known to cross-link DNA that contributes significantly to the cytotoxicity and consequent cell death. Cytosolic NADPH:quinone oxidoreductase 1 (NQO1) and microsomal enzymes have been shown to mediate MMC-induced DNA cross-linking. However, NQO1 plays only a minor role, indicating presence of other cytosolic enzymes/proteins that contribute to this process. In this study, we have characterized a unique cytosolic activity in NQO1-null mice that catalyzed MMC-induced DNA cross-linking. This activity was cofactor independent and dicoumarol insensitive. The unique cytosolic activity was purified to homogeneity. The peptide sequencing of the purified protein identified the unique cytosolic activity as GRP58 (M(r) 58,000 glucose-regulatory protein), also known as GRp57/ER60/ERp61/HIP-70/Q2 and CPT. Immunodepletion of NQO1-null mice liver cytosol and partially purified fractions with anti-GRP58 antibody led to a complete loss of GRP58 protein and consequent significant reduction of MMC-induced DNA cross-linking. Mouse cDNA encoding GRP58 was isolated and sequenced. Chinese hamster ovary cells permanently overexpressing GRP58 showed increased MMC-induced DNA cross-linking and increased cytotoxicity on exposure to MMC. Bacterially expressed and purified GRP58 increased the MMC-induced DNA cross-linking when added to mouse cytosolic samples. A tissue array analysis indicated that GRP58 is ubiquitously expressed among mouse tissues, although at different levels. Expression analysis using matched human tumor/normal array revealed an up-regulation of GRP58 in breast, uterus, lung, and stomach tumors compared with normal tissues of similar origin.

The role of alpha-methylacyl-CoA racemase in bile acid synthesis.

According to current views, the second peroxisomal beta-oxidation pathway is responsible for the degradation of the side chain of bile acid intermediates. Peroxisomal multifunctional enzyme type 2 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(R)-3-hydroxyacyl-CoA dehydrogenase; MFE-2] catalyses the second (hydration) and third (dehydrogenation) reactions of the pathway. Deficiency of MFE-2 leads to accumulation of very-long-chain fatty acids, 2-methyl-branched fatty acids and C(27) bile acid intermediates in plasma, but bile acid synthesis is not blocked completely. In this study we describe an alternative pathway, which allows MFE-2 deficiency to be overcome. The alternative pathway consists of alpha-methylacyl-CoA racemase and peroxisomal multifunctional enzyme type 1 [peroxisomal multifunctional 2-enoyl-CoA hydratase/(S)-3-hydroxyacyl-CoA dehydrogenase; MFE-1]. (24E)-3alpha,7alpha,12alpha-Trihydroxy-5beta-cholest-24-enoyl-CoA, the presumed physiological isomer, is hydrated by MFE-1 with the formation of (24S,25S)-3alpha,7alpha,12alpha,24-tetrahydroxy-5beta-cholestanoyl-CoA [(24S,25S)-24-OH-THCA-CoA], which after conversion by a alpha-methylacyl-CoA racemase into the (24S,25R) isomer can again be dehydrogenated by MFE-1 to 24-keto-3alpha,7alpha,12alpha-trihydroxycholestanoyl-CoA, a physiological intermediate in cholic acid synthesis. The discovery of the alternative pathway of cholesterol side-chain oxidation will improve diagnosis of peroxisomal deficiencies by identification of serum 24-OH-THCA-CoA diastereomer profiles.

The cell biology of MHC class I antigen presentation.

MHC class I antigen presentation refers to the co-ordinated activities of many intracellular pathways that promote the cell surface appearance of MHC class I/beta2m heterodimers loaded with a spectrum of self or foreign peptides. These MHC class I peptide complexes form ligands for CD8 positive T cells and NK cells. MHC class I heterodimers are loaded within the endoplasmic reticulum (ER) with peptides derived from intracellular proteins. Alternatively, MHC class I molecules may be loaded with peptides derived from extracellular proteins in a process called MHC class I cross presentation. This pathway is less well defined but can overlap those pathways operating in classical MHC class I presentation and has recently been reviewed elsewhere (1). This review will address the current concepts regarding the intracellular assembly of MHC class I molecules with their peptide cargo within the ER and their subsequent progress to the cell surface.