Studies on bound trans-4-hydroxy-l-proline in sandal (Santalum album L.)

1. trans-4-Hydroxy-l-proline was found to occur in the bound state in the leaves of sandal (Santalum album L.), in which large amounts of free cis-4-hydroxy-l-proline are also present. 2. Bound trans-4-hydroxy-l-proline was present, associated mainly with a `wall' fraction and a `soluble' fraction roughly in equal proportions. 3. Bound proline is present only in small amounts in the `soluble' fraction but is mostly associated with the `wall' fraction and the other sedimented fractions. 4. In the free amino acid fraction more than 98% of the hydroxyproline had the cis-configuration, whereas in the `wall' and `soluble' fractions more than 90% of the bound hydroxyproline was in the trans-configuration. 5. Various extraction procedures indicated heterogeneity of the hydroxyproline-containing components. Hot 5% (w/v) trichloroacetic acid extracts about 25% of hydroxyproline and m-NaOH extracts an additional 25%. 6. Incorporation of [¹⁴C]proline into the bound hydroxyproline was demonstrated. The hydroxyproline component of the `soluble' fraction does not appear to be the precursor of that of the `wall' fraction.     

Regio- and stereoselective oxygenation of proline derivatives by using microbial 2-oxoglutarate-dependent dioxygenases

We evaluated the substrate specificities of four proline cis-selective hydroxylases toward the efficient synthesis of proline derivatives. In an initial evaluation, 15 proline-related compounds were investigated as substrates. In addition to l-proline and l-pipecolinic acid, we found that 3,4-dehydro-l-proline, l-azetidine-2-carboxylic acid, cis-3-hydroxy-l-proline, and l-thioproline were also oxygenated. Subsequently, the product structures were determined, revealing cis-3,4-epoxy-l-proline, cis-3-hydroxy-l-azetidine-2-carboxylic acid, and 2,3-cis-3,4-cis-3,4-dihydroxy-l-proline.     

Properties, metabolisms, and applications of l-proline analogues

Due to the unique role of l-proline in the folding and structure of protein, a variety of synthetic proline analogues have been developed. l-Proline analogues have been proven to be valuable reagents for studying cellular metabolism and the regulation of macromolecule synthesis in both prokaryotic and eukaryotic cells. In addition to these fundamental researches, they are useful compounds for industrial use. For instance, microorganisms that overproduce l-proline have been obtained by isolating mutants resistant to l-proline analogues. They are also promising candidates for tuning the biological, pharmaceutical, or physicochemical properties of naturally occurring or de novo designed peptides. Among l-proline analogues, l-azetidine-2-carboxylic acid (l-AZC) is a toxic non-proteinogenic amino acid originally found in lily of the valley plants and trans-4-hydroxy-l-proline (4-l-THOP) is the most abundant component of mammalian collagen. Many hydroxyprolines (HOPs), such as 4-l-THOP and cis-4-hydroxy-l-proline (4-l-CHOP), are useful chiral building blocks for the organic synthesis of pharmaceuticals. In addition, l-AZC and 4-l-CHOP, which are potent inhibitors of cell growth, have been tested for their antitumor activity in tissue culture and in vivo. In this review, we describe the recent discoveries regarding the physiological properties and microbial production and metabolism of l-proline analogues, particularly l-AZC and HOPs. Their applications in fundamental research and industrial use are also discussed.     

Novel Enzyme Family Found in Filamentous Fungi Catalyzing trans-4-Hydroxylation of l-Pipecolic Acid

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward l-pipecolic acid (l-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme l-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of l-Pip, and produces optically pure trans-4-hydroxy-l-pipecolic acid (trans-4-l-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had l-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the l and d forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-l-HyPip was successfully developed. Thus, we report a fungal family of l-Pip hydroxylases and the enzymatic preparation of trans-4-l-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities.     

5-Oxoprolinase (l-Pyroglutamate Hydrolase) in Higher Plants: Partial Purification and Characterization of the Wheat Germ Enzyme

5-Oxoprolinase has been found to be widely distributed in higher plants. This enzyme catalyzes the ATP-dependent hydrolysis of 5-oxo-l-proline (l-pyrollidone carboxylate, l-pyroglutamate) to glutamate. The enzyme has been purified almost 60 fold from wheat germ (Triticum aestivum L). This enzyme requires a divalent cation, either Mn²⁺ or Mg²⁺, and a combination of both appears to be the most effective. There is also an absolute requirement for a monovalent cation best fulfilled by either NH₄⁺ or K⁺. The K_m for ATP is 0.4 mm and for 5-oxo-l-proline is 14 μm. A small amount of activity is observed when other purine nucleotides such as ITP and GTP replace ATP. The substitution of the pyrimidine nucleotides CTP and UTP for ATP yield almost completely inactive preparations. The enzyme appears to have an active sulfhydryl group since there is an increase in activity in the presence of dithioerythritol. Preincubation with reagents such as N-ethylmaleimide or iodoacetamide lead to complete inactivation. The presence of this enzyme leads to the speculation of the possible presence of a γ-glutamyl cycle in higher plants.     

Hydroxylation of l-proline to cis-3-hydroxy-l-proline by recombinant Escherichia coli expressing a synthetic l-proline-3-hydroxylase gene

A synthetic gene encoding a Streptomyces l-proline-3-hydroxylase was constructed and used to produce the hydroxylase protein in recombinant Escherichia coli. A fermentation process for growth of this recombinant E. coli for enzyme production was scaled-up to 250 L. A biotransformation process was developed using cell suspensions of the recombinant E. coli and subsequently scaled-up to 10 L for conversion of l-proline to cis-3-hydroxy-l-proline. A reaction yield of 85 M% and d.e. of 99.9% was obtained for cis-3-hydroxy-l-proline.     

Some Effectors on the Biosynthesis of cis-4-Hydroxy-d-proline in Viridogrisein

A system to improve the yield of neoviridogrisein II, in which the cis-4-hydroxy-d-proline is replaced with d-proline, was devised by selective inhibition of the hydroxylation of l-proline with zinc ion in a chemically defined medium. Zinc ion opposed the ferrous ion in the hydroxylation system of Streptomyces griseoviridus P8648.     

Microbial production of N-acetyl cis-4-hydroxy-l-proline by coexpression of the Rhizobium l-proline cis-4-hydroxylase and the yeast N-acetyltransferase Mpr1

The proline analogue cis-4-hydroxy-l-proline (CHOP), which inhibits the biosynthesis of collagen, has been clinically evaluated as an anticancer drug, but its water solubility and low molecular weight limits its therapeutic potential since it is rapidly excreted. In addition, CHOP is too toxic to be practical as an anticancer drug, due primarily to its systematic effects on noncollagen proteins. To promote CHOP’s retention in blood and/or to decrease its toxicity, N-acetylation of CHOP might be a novel approach as a prodrug. The present study was designed to achieve the microbial production of N-acetyl CHOP from l-proline by coexpression of l-proline cis-4-hydroxylases converting l-proline into CHOP (SmP4H) from the Rhizobium Sinorhizobium meliloti and N-acetyltransferase converting CHOP into N-acetyl CHOP (Mpr1) from the yeast Saccharomyces cerevisiae. We constructed a coexpression plasmid harboring both the SmP4H and Mpr1 genes and introduced it into Escherichia coli BL21(DE3) or its l-proline oxidase gene-disrupted (ΔputA) strain. M9 medium containing l-proline produced more N-acetyl CHOP than LB medium containing l-proline. E. coli ΔputA cells accumulated l-proline (by approximately 2-fold) compared to that in wild-type cells, but there was no significant difference in CHOP production between wild-type and ΔputA cells. The addition of NaCl and l-ascorbate resulted in a 2-fold increase in N-acetyl CHOP production in the l-proline-containing M9 medium. The highest yield of N-acetyl CHOP was achieved at 42 h cultivation in the optimized medium. Five unknown compounds were detected in the total protein reaction, probably due to the degradation of N-acetyl CHOP. Our results suggest that weakening of the degradation or deacetylation pathway improves the productivity of N-acetyl CHOP.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

The Aspergillus nidulans Proline Permease as a Model for Understanding the Factors Determining Substrate Binding and Specificity of Fungal Amino Acid Transporters

Amino acid uptake in fungi is mediated by general and specialized members of the yeast amino acid transporter (YAT) family, a branch of the amino acid polyamine organocation (APC) transporter superfamily. PrnB, a highly specific l-proline transporter, only weakly recognizes other Put4p substrates, its Saccharomyces cerevisiae orthologue. Taking advantage of the high sequence similarity between the two transporters, we combined molecular modeling, induced fit docking, genetic, and biochemical approaches to investigate the molecular basis of this difference and identify residues governing substrate binding and specificity. We demonstrate that l-proline is recognized by PrnB via interactions with residues within TMS1 (Gly⁵⁶, Thr⁵⁷), TMS3 (Glu¹³⁸), and TMS6 (Phe²⁴⁸), which are evolutionary conserved in YATs, whereas specificity is achieved by subtle amino acid substitutions in variable residues. Put4p-mimicking substitutions in TMS3 (S130C), TMS6 (F252L, S253G), TMS8 (W351F), and TMS10 (T414S) broadened the specificity of PrnB, enabling it to recognize more efficiently l-alanine, l-azetidine-2-carboxylic acid, and glycine without significantly affecting the apparent K_m for l-proline. S253G and W351F could transport l-alanine, whereas T414S, despite displaying reduced proline uptake, could transport l-alanine and glycine, a phenotype suppressed by the S130C mutation. A combination of all five Put4p-ressembling substitutions resulted in a functional allele that could also transport l-alanine and glycine, displaying a specificity profile impressively similar to that of Put4p. Our results support a model where residues in these positions determine specificity by interacting with the substrates, acting as gating elements, altering the flexibility of the substrate binding core, or affecting conformational changes of the transport cycle.     

Bacterial catabolism of threonine. Threonine degradation initiated by l-threonine acetaldehyde-lyase (aldolase) in species of Pseudomonas

1. The route of l-threonine degradation was studied in four strains of the genus Pseudomonas able to grow on the amino acid and selected because of their high l-threonine aldolase activity. Growth and manometric results were consistent with the cleavage of l-threonine to acetaldehyde+glycine and their metabolism via acetate and serine respectively. 2. l-Threonine aldolases in these bacteria exhibited pH optima in the range 8.0–8.7 and K_m values for the substrate of 5–10mm. Extracts exhibited comparable allo-l-threonine aldolase activities, K_m values for this substrate being 14.5–38.5mm depending on the bacterium. Both activities were essentially constitutive. Similar activity ratios in extracts, independent of growth conditions, suggested a single enzyme. The isolate Pseudomonas D2 (N.C.I.B. 11097) represents the best source of the enzyme known. 3. Extracts of all the l-threonine-grown pseudomonads also possessed a CoA-independent aldehyde dehydrogenase, the synthesis of which was induced, and a reversible alcohol dehydrogenase. The high acetaldehyde reductase activity of most extracts possibly resulted in the underestimation of acetaldehyde dehydrogenase. 4. l-Serine dehydratase formation was induced by growth on l-threonine or acetate+glycine. Constitutively synthesized l-serine hydroxymethyltransferase was detected in extracts of Pseudomonas strains D2 and F10. The enzyme could not be detected in strains A1 and N3, probably because of a highly active `formaldehyde-utilizing' system. 5. Ion-exchange and molecular exclusion chromatography supported other evidence that l-threonine aldolase and allo-l-threonine aldolase activities were catalysed by the same enzyme but that l-serine hydroxymethyltransferase was distinct and different. These results contrast with the specificities of some analogous enzymes of mammalian origin.     

The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

1. all-trans-Retinoic acid at concentrations greater than 10⁻⁷m stimulated the incorporation of d-[³H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl₂-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[³H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10⁻⁵m-all-trans-retinoic acid. The labelling of other epidermal components with d-[³H]glucosamine, ³⁵SO₄²⁻, l-[¹⁴C]fucose and U-¹⁴C-labelled l-amino acids was not significantly affected by 10⁻⁵m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[³H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[³H]-glucosamine into extracellular epidermal glycosaminoglycans in 10⁻⁵m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10⁻⁵m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10⁻⁵m-all-trans-retinoic acid-treated epidermis. 7. The labelling of epidermal plasma membranes isolated from d-[³H]glucosamine-labelled skin slices by sucrose density gradient centrifugation was similar in control and 10⁻⁵m-all-trans-retinoic acid-treated tissue. 8. The results indicate that increased synthesis of mainly extracellular glycosaminoglycans (largely hyaluronic acid) may be the first response of the epidermis to excess all-trans-retinoic acid.     

Biosynthesis of Arabinogalactan-Protein in Lolium multiflorum (Ryegrass) Endosperm Cells : III. Subcellular Distribution of Prolyl Hydroxylase

The peptidyl prolyl hydroxylase responsible for the formation of hydroxyproline during arabinogalactan-protein biosynthesis in Lolium multiflorum (ryegrass) endosperm cells is a membrane-associated enzyme which will catalyze the hydroxylation of poly(l-proline) in the presence of oxygen, α-ketoglutarate, ferrous ion, and ascorbate. The K_m for poly(l-proline) (8000 molecular weight) is 40 micromolar. The enzyme will also hydroxylate the protocollagen analog (Pro-Pro-Gly)₅·4H₂O.     

New Amides from Buckwheat Seeds (Fagopyrum esculentum Moench)

Two new amino acid amides which yield in acid hydrolysis isomeric hydroxybenzylamines and amino acids have been isolated from the achenes of Fagopyrum esculentum Moench. One of them called BN-II is composed of salicylamine and allo-4-hydroxy-l-glutamic acid, and the other, BN-III, p-hydroxybenzylamine and l-glutamic acid. These coupled compounds link one another to form an amide respectively. Finally the structures of BN-II and BN-III were determined to be N⁵-(2′-hydroxybenzyl)-allo-4-hydroxy-l-glutamine and N⁵-(4′-hydroxybenzyl)-l-glutamine respectively from their chemical and spectrometry properties.     

Reversal of hydroxyproline-induced inhibition of elongation of Avena coleoptiles

Hydroxy-l-proline-induced inhibition of elongation of Avena coleoptile segments was measured in water and in indole-3-acetic acid. This inhibition was completely reversed by l-proline.     

One-Pot Production of l-threo-3-Hydroxyaspartic Acid Using Asparaginase-Deficient Escherichia coli Expressing Asparagine Hydroxylase of Streptomyces coelicolor A3(2)

We developed a novel process for efficient synthesis of l-threo-3-hydroxyaspartic acid (l-THA) using microbial hydroxylase and hydrolase. A well-characterized mutant of asparagine hydroxylase (AsnO-D241N) and its homologous enzyme (SCO2693-D246N) were adaptable to the direct hydroxylation of l-aspartic acid; however, the yields were strictly low. Therefore, the highly stable and efficient wild-type asparagine hydroxylases AsnO and SCO2693 were employed to synthesize l-THA. By using these recombinant enzymes, l-THA was obtained by l-asparagine hydroxylation by AsnO followed by amide hydrolysis by asparaginase via 3-hydroxyasparagine. Subsequently, the two-step reaction was adapted to one-pot bioconversion in a test tube. l-THA was obtained in a small amount with a molar yield of 0.076% by using intact Escherichia coli expressing the asnO gene, and thus, two asparaginase-deficient mutants of E. coli were investigated. A remarkably increased l-THA yield of 8.2% was obtained with the asparaginase I-deficient mutant. When the expression level of the asnO gene was enhanced by using the T7 promoter in E. coli instead of the lac promoter, the l-THA yield was significantly increased to 92%. By using a combination of the E. coli asparaginase I-deficient mutant and the T7 expression system, a whole-cell reaction in a jar fermentor was conducted, and consequently, l-THA was successfully obtained from l-asparagine with a maximum yield of 96% in less time than with test tube-scale production. These results indicate that asparagine hydroxylation followed by hydrolysis would be applicable to the efficient production of l-THA.     

Cytokinins in tRNA Obtained from Spinacia oleracea L. Leaves and Isolated Chloroplasts

Cytokinin-active ribonucleosides have been isolated from tRNA of whole spinach (Spinacia oleracea L.) leaves and isolated spinach chloroplasts. The tRNA from spinach leaf blades contained: 6-(4-hydroxy-3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine (cis and trans isomers), 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine, and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (cis and trans isomers). A method for isolation of large amounts of intact chloroplasts was developed and subsequently used for the isolation of chloroplast tRNA. The chloroplast tRNA contained 6-(3-methyl-2-butenylamino)-9-β-d-ribofuranosylpurine and 6-(4-hydroxy-3-methyl-2-butenylamino)-2-methylthio-9-β-d -ribofuranosylpurine (the cis isomer only). The structures of these compounds were assigned on the basis of their chromatographic properties and mass spectra of trimethylsilyl derivatives which were identical with those of the corresponding synthetic compounds. The results of this study indicate that ribosylzeatin was present in spinach leaf tRNA, but absent from the purified chloroplast tRNA preparation.