Biosynthesis of trans-4-hydroxy-L-proline by Streptomyces griseoviridus.

Radioisotopic experiments have revealed that free trans-4-hydroxy-L-proline is an intermediate synthesized from L-proline during formation of the peptide-bound cis-4-hydroxy-D-proline residue in the antibiotic, etamycin. This conclusion was based on the fact that 1) both radiolabeled L-proline and trans-4-hydroxy-L-proline are precursors of the bound D-imino acid as noted previously by Hook and Vining ((1973) J. Chem. Soc. Chem. Commun. 185-186; (1973) Can. J. Biochem. 51, 1630-1637), 2) the unlabeled trans isomer specifically inhibited the incorporation of radiolabel from proline into the antibiotic, 3) the 14C-hydroxyimino-acid was isolated from the intracellular pool and medium following incubations with L-[14C]proline during antibiotic biosynthesis and when etamycin synthesis was blocked by D-leucine. By means of chromatographic and enzymatic analyses, it was established that the free imino acid possesses the trans-L configuration.     

Proline accumulation in a barley mutant resistant to trans-4-hydroxy-L-proline

Five proline analogues were tested for inhibition of the growth of mature barley (Hordeum vulgare L.) embryos in sterile culture. Inhibition by all analogues was relieved by proline. Inhibition by trans-4-hydroxy-L-proline was relieved by low amounts of proline. Twenty thousand mature embryos were dissected from M₂ seeds after sodium azide mutagenesis. Four plants (Rothamsted 5201, 6102, 6901, 6902) were selected with good growth on 4 mM trans-4-hydroxyproline. Properties of mutant R5201 were studied in detail. Selfed progeny of R5201 were all resistant to trans-4-hydroxyproline and also to L-thiazolidine-4-carboxylic acid and trans-3-hydroxy-L-proline but not L-azetidine-2-carboxylic acid. The content of soluble proline in progeny of R5201 was higher in leaves by a factor of up to six-fold. Proline content was measured in the soluble fraction of the terminal 20 mm of 4 d old plants subjected to severe water stress in 40% w/v polyethylene glycol. Leaves of the mutant contained more proline initially and accumulated proline morer rapidly than the parental leaves. As mutant leaves were larger and lost water more rapidly the greater increase in proline may have been caused by more severe water stress. Resistance to trans-4-hydroxyproline in R5201 was due to a single partially dominant nuclear gene.Abbreviations AZC L-azetidine-2-carboxylic acid - HYP trans-4-hydroxy-L-proline - ORN L-ornithine - CIT L-citrulline     

Isosteric phosphonate pyrrolidine-based dinucleoside monophosphate analogues

A novel alpha- and beta-configured pyrrolidine nucleoside phosphonates in adenine series were synthesized from trans-4-hydroxy-L-proline as starting material. d(ApA) analogues were also prepared and studied with respect to their hybridization properties with polyU.     

Identification of a human trans-3-hydroxy-L-proline dehydratase, the first characterized member of a novel family of proline racemase-like enzymes

A family of eukaryotic proline racemase-like genes has recently been identified. Several members of this family have been well characterized and are known to catalyze the racemization of free proline or trans-4-hydroxyproline. However, the majority of eukaryotic proline racemase-like proteins, including a human protein called C14orf149, lack a specific cysteine residue that is known to be critical for racemase activity. Instead, these proteins invariably contain a threonine residue at this position. The function of these enzymes has remained unresolved until now. In this study, we demonstrate that three enzymes of this type, including human C14orf149, catalyze the dehydration of trans-3-hydroxy-L-proline to Δ(1)-pyrroline-2-carboxylate (Pyr2C). These are the first enzymes of this subclass of proline racemase-like genes for which the enzymatic activity has been resolved. C14orf149 is also the first human enzyme that acts on trans-3-hydroxy-L-proline. Interestingly, a mutant enzyme in which the threonine in the active site is mutated back into cysteine regained 3-hydroxyproline epimerase activity. This result suggests that the enzymatic activity of these enzymes is dictated by a single residue. Presumably, human C14orf149 serves to degrade trans-3-hydroxy-L-proline from the diet and originating from the degradation of proteins that contain this amino acid, such as collagen IV, which is an important structural component of basement membrane.     

Stereoselective O-glycosylation of trans-4-hydroxy-L-proline derivatives promoted by silver zeolite

Trans-4-hydroxy-L-proline has been converted to four imino- and carboxyl-blocked derivatives which are suitable for the synthesis of 4-O-glycosyl conjugates. Reaction of these derivatives with 2,3,5-tri-O-benzyl-alpha-L-arabinofuranosyl chloride in the presence of a silver zeolite promoter yielded the blocked beta-furanosyl amino-acid conjugates. Deprotection gave trans-4-(beta-L-arabinofuranosyloxy)-L-proline which was characterised as its crystalline isopropyl ester. 13C-NMR Data are presented for the compounds described.     

Structural pre-organization of peptide nucleic acids

Introduction of constraint via chemical bridging in the aegPNA leads to the five or six membered cyclic structures that may contribute towards maintaining the balance between rigidity and flexibility of the PNA backbone. The significant promise of our approach to use the naturally occurring trans-4-hydroxy-L-proline to arrive at different chirally pure cyclic PNA analogs and their DNA binding properties will be presented.     

Microbial proline 4-hydroxylase screening and gene cloning.

Microbial proline 4-hydroxylases, which hydroxylate free L-proline to trans-4-hydroxy-L-proline, were screened in order to establish an industrial system for biotransformation of L-proline to trans-4-hydroxy-L-proline. Enzyme activities were detected in eight strains, including strains of Dactylosporangium spp. and Amycolatopsis spp. The Dactylosporangium sp. strain RH1 enzyme was partially purified 3,300-fold and was estimated to be a monomer polypeptide with an apparent molecular mass of 31 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Degenerate primers based on the N-terminal amino acid sequence of the 31-kDa polypeptide were synthesized in order to amplify the corresponding 71-bp DNA fragment. A 5.5-kbp DNA fragment was isolated by using the 71-bp fragment labeled with digoxigenin as a probe for a genomic library of Dactylosporangium sp. strain RH1 constructed in Escherichia coli. One of the open reading frames found in the cloned DNA, which encoded a 272-amino-acid polypeptide (molecular mass, 29, 715 daltons), was thought to be a proline 4-hydroxylase gene. The gene was expressed in E. coli as a fused protein with the N-terminal 34 amino acids of the beta-galactosidase alpha-fragment. The E. coli recombinant exhibited proline 4-hydroxylase activity that was 13. 6-fold higher than the activity in the original strain, Dactylosporangium sp. strain RH1. No homology was detected with other 2-oxoglutarate-dependent dioxygenases when databases were searched; however, the histidine motif conserved in 2-oxoglutarate-dependent dioxygenases was found in the gene.     

Phosphono peptide nucleic acids with a constrained hydroxyproline-based backbone

DNA mimics representing negatively charged analogues of peptide nucleic acids (PNAs), particularly hetero-oligomers constructed from alternating phosphono-PNA residues (pPNA) and monomers on the base of trans-4-hydroxy-L-proline (HypNA) as well as mimics composed of phosphono-HypNA monomers (pHypNA) were tested in a set of in vitro and in vivo assays, and they demonstrated a high potential for the use in nucleic acid based diagnostic, isolation of nucleic acids and antisense experiments.     

PNA-related oligonucleotide mimics and their evaluation for nucleic acid hybridization studies and analysis

DNA mimics containing phosphonate analogues of PNAs (pPNAs), particularly PNA-pPNA hybrids as well as hetero-oligomers consisted of pPNA units and PNA-like molecules on the base of trans-4-hydroxy-L-proline (HypNA) have been synthesized. The evaluation of their effectiveness in assays based on the hybridization technique in the comparison with natural oligonucleotides and classical PNAs has shown a high potential of these mimics as sensor molecules for nucleic acid based diagnostics and as molecular probes for mRNA isolation.     

Messenger RNA isolation using novel PNA analogues

Homo-Thy hetero-oligomer probes composed of trans-4-hydroxy-L-proline based PNA-like (HypNA) monomers and phosphono PNA (pPNA) monomers demonstrated strong binding to complementary poly A+ RNA strands. We used a mixture of chimeric oligomers containing both "linear" and "clamping" PNA-analogues to develop an mRNA isolation procedure and demonstrate the improved recovery of RNA molecules with secondary structure at the 3'end as well as RNAs with short poly A tails.     

Synthesis and pharmacochemical study of novel polyfunctional molecules combining anti-inflammatory, antioxidant, and hypocholesterolemic properties

We have designed and synthesized a series of novel molecules having a residue of a classical NSAID and an antioxidant moiety, both attached through amide bonds to a known nootropic structure, an L-proline, trans-4-hydroxy-L-proline or DL-pipecolinic acid residue. The compounds were found to retain anti-inflammatory and antioxidant activities, to acquire hypocholesterolemic action, and to possess a greatly reduced gastrointestinal toxicity. The novel molecules could find useful applications, among others, in slowing the progression or delaying the onset of neurodegenerative diseases.     

Nouveaux acides amines libres de Afzelia bella: trans-hydroxy-4-L-proline et trans-carboxy-4-L-proline

Two new free amino acids have been isolated from seeds of Afzelia bella: trans-4-hydroxy-L-proline and trans-4-carboxy-L-proline. They are accompanied by relatively large quantities of proline, pipecolic, acid, 4-methylene-DL-proline, cis-4-hydroxymethyl-L-proline and 4-methylene-L-glutamic acid. Small amounts of acetylornithine are also present. It is the first time that trans-4-hydroxy-L-proline has been found as a component of the free amino pool of plants.     

Design, synthesis, and preliminary evaluation of 4-(6-(3-nitroguanidino)hexanamido)pyrrolidine derivatives as potential iNOS inhibitors

A series of 4-(6-(3-nitroguanidino)hexanamido)pyrrolidine derivatives were synthesized and evaluated for their abilities to inhibit inducible nitric oxide synthase (iNOS) isoform. All target compounds were prepared in 11 steps from commercially trans-4-hydroxy-L-proline. The preliminary pharmacological test showed that three compounds, 17, 21, and 30, have the good potency (IC(50)=2.36, 2.68, 2.5 microM, respectively) which are compared to the NOS inhibitor N(G)-nitroarginine(L-NNA) (IC(50)=14.74 microM), and could be used as lead compounds for exploring new iNOS inhibitors in the future.     

Solubilization and Purification of Rat Liver Microsomal Trans-2-Enoyl-CoA Hydratase

It is well known that fatty acid chain elongation involves four steps : (a) condensation of a primer with malonyl-CoA to form the β-keto acyl CoA; (b) reduction of the resulting β -keto acyl CoA to a secondary alcohol, β-hydroxyl acyl CoA; (c) dehydration of the alcohol to form trans-2-enoyl CoA; and (d) reduction of the trans-2-enoyl CoA to give an acid 2 carbon atoms longer than the primer. ^1–2The enzyme involved in step (c) is trans-2-enoyl-CoA hydratase, It can perform a reversible hydration of α, β-unsaturated fatty acid chain elongation. ³A partial purification of this enzyme has been reported by Bernert and Sprecher. ⁴The purification of enoyl-CoA hydratase from mycobacterium smegmatis ⁵and from rat mitochondria have also been reported; ⁶however, the purification of enoyl-hydratase from rat liver has not been reported.     

Enzymatic preparation of 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline from (α-N-protected)-L-ornithine using a transaminase or an amine oxidase

N-Cbz-4,5-dehydro-L-prolineamide or N-Boc-4,5-dehydro-L-prolineamide are alternative key intermediates for the synthesis of saxagliptin, a dipeptidyl peptidase IV (DPP4) inhibitor recently approved for treatment of type 2 diabetes mellitus. An efficient biocatalytic method was developed for conversion of L-ornithine, N-α-benzyloxycarbonyl (Cbz)-L-ornthine, and N-α-tert-butoxycarbonyl (Boc)-L-ornithine to 5-hydroxy-L-proline, N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline, respectively. Rec. Escherichia coli expressing lysine-ε-aminotransferase and rec Pichia pastoris expressing L-ornithine oxidase were used for these conversions. N-Cbz-5-hydroxy-L-proline, and N-Boc-5-hydroxy-L-proline were chemically converted to key intermediates N-Cbz-4,5-dehydro-L-prolineamide and N-Boc-4,5-dehydro-L-prolineamide, respectively.     

Elucidation of the Mechanism by Which 9-(Trans-2′, Trans-3′-Dihydroxycyclopent-4′-enyl)-Adenine Lnactivates S-Adenosylhomocysteine Hydrolase and Elevates Cellular Levels of Sadenosylhomocysteinew

Abstract

Evidence is presented that 9-(trans-2′, trans-3′-dihydroxycyclopent-4′-enyl)-adenine (1) is oxidized to the 3′-ketonucleoside 2 with concomitant reduction of NAD⁺ to NADH by S-adenosylhomcysteine hydrolase. We also describe how the carbocyclic nucleoside I has been used to reveal the metabolic relationships between Sadenosylmethionine, S-adenosylhomocysteine and homocystcine in murine L-929 cells.     

Inhibition of angiotensin converting enzyme by L-alanyl-4 or 5-substituted-L-prolines and their N alpha-phosphoryl-derivatives

A series of L-alanyl-4 or 5-substituted L-prolines, such as L-alanyl-L-thiazolidine-4-carboxylic acid, L-alanyl-5-oxo-L-proline, L-alanyl-trans-4-hydroxy-L-proline and L-alanyl-cis-4-hydroxy-L-proline as well as their corresponding N alpha-phosphoryl derivatives, were synthesized and studied for inhibition against angiotensin converting enzyme. Furanacryloyl-phenylalanyl-glycyl-glycine was used as substrate in 50 mM Tris hydrochloride buffer at pH 7.5 containing 1 microM zinc acetate. N alpha-Phosphoryl-L-alanyl-L-thiazolidine-4-carboxylic acid and N alpha-phosphoryl-L-alanyl-trans-4-hydroxy-L-proline competitively inhibit angiotensin converting enzyme with Ki values of 68 microM and 89.3 microM, respectively. Smaller inhibition against angiotensin converting enzyme was obtained with the rest of compounds studied here.     

Characterization of cis-4-hydroxy-D-proline dehydrogenase from Sinorhizobium meliloti

The hypO gene from Sinorhizobium meliloti, located within the trans-4-hydroxy-L-proline metabolic gene cluster, was first successfully expressed in the host Pseudomonas putida. Purified HypO protein functioned as a FAD-containing cis-4-hydroxy-D-proline dehydrogenase with a homomeric structure. In contrast to other known enzymes, significant activity for D-proline was found, confirming a previously proposed potential involvement in D-proline metabolism.     

Trans-α-xylosidase,a widespread enzyme activity in plants,introduces (1→4)-α-d-xylobiose side-chains into xyloglucan structures

Angiosperms possess a retaining trans-α-xylosidase activity that catalyses the inter-molecular transfer of xylose residues between xyloglucan structures. To identify the linkage of the newly transferred α-xylose residue, we used [Xyl-³H]XXXG (xyloglucan heptasaccharide) as donor substrate and reductively-aminated xyloglucan oligosaccharides (XGO–NH₂) as acceptor. Asparagus officinalis enzyme extracts generated cationic radioactive products ([³H]Xyl·XGO–NH₂) that were Driselase-digestible to a neutral trisaccharide containing an α-[³H]xylose residue. After borohydride reduction, the trimer exhibited high molybdate-affinity, indicating xylobiosyl-(1→6)-glucitol rather than a di-xylosylated glucitol. Thus the trans-α-xylosidase had grafted an additional α-[³H]xylose residue onto the xylose of an isoprimeverose unit. The trisaccharide was rapidly acetolysed to an α-[³H]xylobiose, confirming the presence of an acetolysis-labile (1→6)-bond. The α-[³H]xylobiitol formed by reduction of this α-[³H]xylobiose had low molybdate-affinity, indicating a (1→2) or (1→4) linkage. In NaOH, the α-[³H]xylobiose underwent alkaline peeling at the moderate rate characteristic of a (1→4)-disaccharide. Finally, we synthesised eight non-radioactive xylobioses [α and β; (1↔1), (1→2), (1→3) and (1→4)] and found that the [³H]xylobiose co-chromatographed only with (1→4)-α-xylobiose. We conclude that Asparagus trans-α-xylosidase activity generates a novel xyloglucan building block, α-d-Xylp-(1→4)-α-d-Xylp-(1→6)-d-Glc (abbreviation: ‘V’). Modifying xyloglucan structures in this way may alter oligosaccharin activities, or change their suitability as acceptor substrates for xyloglucan endotransglucosylase (XET) activity.     

Isosteric Phosphonate Pyrrolidine-Based Dinucleoside Monophosphate Analogues

Abstract

A novel α- and β-configured pyrrolidine nucleoside phosphonates in adenine series were synthesized from trans-4-hydroxy-L-proline as starting material. d(ApA) analogues were also prepared and studied with respect to their hybridization properties with polyU.