Nonenzymatic Cyclization Research Articles

A Role in 15-Deacetylcalonectrin Acetylation in the Non-Enzymatic Cyclization of an Earlier Bicyclic Intermediate in Fusarium Trichothecene Biosynthesis.

The trichothecene biosynthesis in Fusarium begins with the cyclization of farnesyl pyrophosphate to trichodiene, followed by subsequent oxygenation to isotrichotriol. This initial bicyclic intermediate is further cyclized to isotrichodermol (ITDmol), a tricyclic precursor with a toxic trichothecene skeleton. Although the first cyclization and subsequent oxygenation are catalyzed by enzymes encoded by Tri5 and Tri4, the second cyclization occurs non-enzymatically. Following ITDmol formation, the enzymes encoded by Tri101, Tri11, Tri3, and Tri1 catalyze 3-O-acetylation, 15-hydroxylation, 15-O-acetylation, and A-ring oxygenation, respectively. In this study, we extensively analyzed the metabolites of the corresponding pathway-blocked mutants of Fusarium graminearum. The disruption of these Tri genes, except Tri3, led to the accumulation of tricyclic trichothecenes as the main products: ITDmol due to Tri101 disruption; a mixture of isotrichodermin (ITD), 7-hydroxyisotrichodermin (7-HIT), and 8-hydroxyisotrichodermin (8-HIT) due to Tri11 disruption; and a mixture of calonectrin and 3-deacetylcalonectrin due to Tri1 disruption. However, the ΔFgtri3 mutant accumulated substantial amounts of bicyclic metabolites, isotrichotriol and trichotriol, in addition to tricyclic 15-deacetylcalonectrin (15-deCAL). The ΔFgtri5ΔFgtri3 double gene disruptant transformed ITD into 7-HIT, 8-HIT, and 15-deCAL. The deletion of FgTri3 and overexpression of Tri6 and Tri10 trichothecene regulatory genes did not result in the accumulation of 15-deCAL in the transgenic strain. Thus, the absence of Tri3p and/or the presence of a small amount of 15-deCAL adversely affected the non-enzymatic second cyclization and C-15 hydroxylation steps.

Chromatographic measurement of 3-hydroxyanthranilate 3,4-dioxygenase activity reveals that edaravone can mitigate the formation of quinolinic acid through a direct enzyme inhibition

Herein it is reported the development and application of two chromatographic assays for the measurement of the activity of 3-Hydroxyanthranilate-3,4-dioxygenase (3HAO). Such an enzyme converts 3-Hydroxyanthranilic acid (3HAA) to 2-amino-3-carboxymuconic semialdehyde (ACMS), which undergo a spontaneous, non-enzymatic cyclization to produce quinolinic acid (QUIN). The enzyme activity was measured by quantitation of the substrate consumption over time either with spectrophotometric (UV) or mass spectrometric (MS) detection upon reversed-phase chromatographic separation. MS detection resulted more selective and sensitive, but less accurate and precise. However, both methods have sufficient sensitivity to allow the measurement of enzyme activity with consistent results compared to literature data. Since MS detection allowed less sample consumption it was used to calculate the kinetics parameters (i.e., Vmax and Kd) of recombinant 3HAO. Another MS-based method was then developed to measure the amount of QUIN produced, revealing an incomplete conversion of 3HAA to QUIN. As suggested by previous studies, the enzyme activity was apparently sensitive to the redox state of the enzyme thiols. In fact, thiol reducing agents such as dithiothreitol (DTT) and glutathione (GSH), can alter the enzyme activity although the investigation on the exact mechanism involved in such effect was beyond the scope of the research. Interestingly, edaravone (EDA) induced an in vitro suppression of QUIN production through direct, competitive 3HAO inhibition. EDA is a molecule approved for the treatment of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease associated with an increase of QUIN concentrations in both serum and cerebrospinal fluid. Although EDA was reported to mitigate ALS progression its mode of action is still largely unknown. Some studies reported antioxidant and radical scavenger properties of EDA, but none confirm a direct activity as 3HAO enzyme inhibitor. Since QUIN is reported to be a neurotoxic metabolite, 3HAO inhibition can contribute to the beneficial effect of EDA in ALS, although such a mechanism must be then confirmed in vivo. However, EDA might be a convenient scaffold for the design of selective 3HAO inhibitors with potential applications in ALS treatment.

Alcohol Dehydrogenase Activity Converts 3″-Hydroxy-geranylhydroquinone to an Aldehyde Intermediate for Shikonin and Benzoquinone Derivatives in Lithospermum erythrorhizon.

Shikonin derivatives are red naphthoquinone pigments produced by several boraginaceous plants, such as Lithospermum erythrorhizon. These compounds are biosynthesized from p-hydroxybenzoic acid and geranyl diphosphate. The coupling reaction that yields m-geranyl-p-hydroxybenzoic acid has been actively characterized, but little is known about later biosynthetic reactions. Although 3″-hydroxy-geranylhydroquinone produced from geranylhydroquinone by CYP76B74 has been regarded as an intermediate of shikonin derivatives, the next intermediate has not yet been identified. This study describes a novel alcohol dehydrogenase activity in L. erythrorhizon cell cultures. This enzyme was shown to oxidize the 3″-alcoholic group of (Z)-3″-hydroxy-geranylhydroquinone to an aldehyde moiety concomitant with the isomerization at the C2'-C3' double bond from the Z-form to the E-form. An enzyme oxidizing this substrate was not detected in other plant cell cultures, suggesting that this enzyme is specific to L. erythrorhizon. The reaction product, (E)-3″-oxo-geranylhydroquinone, was further converted to deoxyshikonofuran, another meroterpenoid metabolite produced in L. erythrorhizon cells. Although nonenzymatic cyclization occurred slowly, it was more efficient in the presence of crude enzymes of L. erythrorhizon cells. This activity was detected in both shikonin-producing and nonproducing cells, suggesting that the aldehyde intermediate at the biosynthetic branch point between naphthalene and benzo/hydroquinone ring formation likely constitutes a key common intermediate in the synthesis of shikonin and benzoquinone products, respectively.

Efficient nonenzymatic cyclization and domain shuffling drive pyrrolopyrazine diversity from truncated variants of a fungal NRPS

Nonribosomal peptide synthetases (NRPSs) generate the core peptide scaffolds of many natural products. These include small cyclic dipeptides such as the insect feeding deterrent peramine, which is a pyrrolopyrazine (PPZ) produced by grass-endophytic Epichloë fungi. Biosynthesis of peramine is catalyzed by the 2-module NRPS, PpzA-1, which has a C-terminal reductase (R) domain that is required for reductive release and cyclization of the NRPS-tethered dipeptidyl-thioester intermediate. However, some PpzA variants lack this R domain due to insertion of a transposable element into the 3' end of ppzA We demonstrate here that these truncated PpzA variants utilize nonenzymatic cyclization of the dipeptidyl thioester to a 2,5-diketopiperazine (DKP) to synthesize a range of novel PPZ products. Truncation of the R domain is sufficient to subfunctionalize PpzA-1 into a dedicated DKP synthetase, exemplified by the truncated variant, PpzA-2, which has also evolved altered substrate specificity and reduced N-methyltransferase activity relative to PpzA-1. Further allelic diversity has been generated by recombination-mediated domain shuffling between ppzA-1 and ppzA-2, resulting in the ppzA-3 and ppzA-4 alleles, each of which encodes synthesis of a unique PPZ metabolite. This research establishes that efficient NRPS-catalyzed DKP biosynthesis can occur in vivo through nonenzymatic dipeptidyl cyclization and presents a remarkably clean example of NRPS evolution through recombinant exchange of functionally divergent domains. This work highlights that allelic variants of a single NRPS can result in a surprising level of secondary metabolite diversity comparable to that observed for some gene clusters.

The killer of Socrates: Coniine and Related Alkaloids in the Plant Kingdom.

Coniine, a polyketide-derived alkaloid, is poisonous to humans and animals. It is a nicotinic acetylcholine receptor antagonist, which leads to inhibition of the nervous system, eventually causing death by suffocation in mammals. Coniine’s most famous victim is Socrates who was sentenced to death by poison chalice containing poison hemlock in 399 BC. In chemistry, coniine holds two historical records: It is the first alkaloid the chemical structure of which was established (in 1881), and that was chemically synthesized (in 1886). In plants, coniine and twelve closely related alkaloids are known from poison hemlock (Conium maculatum L.), and several Sarracenia and Aloe species. Recent work confirmed its biosynthetic polyketide origin. Biosynthesis commences by carbon backbone formation from butyryl-CoA and two malonyl-CoA building blocks catalyzed by polyketide synthase. A transamination reaction incorporates nitrogen from l-alanine and non-enzymatic cyclization leads to γ-coniceine, the first hemlock alkaloid in the pathway. Ultimately, reduction of γ-coniceine to coniine is facilitated by NADPH-dependent γ-coniceine reductase. Although coniine is notorious for its toxicity, there is no consensus on its ecological roles, especially in the carnivorous pitcher plants where it occurs. Lately there has been renewed interest in coniine’s medical uses particularly for pain relief without an addictive side effect.

A Simple Defined Medium for the Production of True Diketopiperazines in Xylella fastidiosa and Their Identification by Ultra-Fast Liquid Chromatography-Electrospray Ionization Ion Trap Mass Spectrometry.

Diketopiperazines can be generated by non-enzymatic cyclization of linear dipeptides at extreme temperature or pH, and the complex medium used to culture bacteria and fungi including phytone peptone and trypticase peptone, can also produce cyclic peptides by heat sterilization. As a result, it is not always clear if many diketopiperazines reported in the literature are artifacts formed by the different complex media used in microorganism growth. An ideal method for analysis of these compounds should identify whether they are either synthesized de novo from the products of primary metabolism and deliver true diketopiperazines. A simple defined medium (X. fastidiosa medium or XFM) containing a single carbon source and no preformed amino acids has emerged as a method with a particularly high potential for the grown of X. fastidiosa and to produce genuine natural products. In this work, we identified a range of diketopiperazines from X. fastidiosa 9a5c growth in XFM, using Ultra-Fast Liquid Chromatography coupled with mass spectrometry. Diketopiperazines are reported for the first time from X. fastidiosa, which is responsible for citrus variegated chlorosis. We also report here fatty acids from X. fastidiosa, which were not biologically active as diffusible signals, and the role of diketopiperazines in signal transduction still remains unknown.

The bacterial catabolism of polycyclic aromatic hydrocarbons: Characterization of three hydratase-aldolase-catalyzed reactions

Summary Polycyclic aromatic hydrocarbons (PAHs) are highly toxic, pervasive environmental pollutants with mutagenic, teratogenic, and carcinogenic properties. There is interest in exploiting the nutritional capabilities of microbes to remove PAHs from various environments including those impacted by improper disposal or spills. Although there is a considerable body of literature on PAH degradation, the substrates and products for many of the enzymes have never been identified and many proposed activities have never been confirmed. This is particularly true for high molecular weight PAHs (e.g., phenanthrene, fluoranthene, and pyrene). As a result, pathways for the degradation of these compounds are proposed to follow one elucidated for naphthalene with limited experimental verification. In this pathway, ring fission produces a species that can undergo a non-enzymatic cyclization reaction. An isomerase opens the ring and catalyzes a cis to trans double bond isomerization. The resulting product is the substrate for a hydratase-aldolase, which catalyzes the addition of water to the double bond of an α,β-unsaturated ketone, followed by a retro-aldol cleavage. Initial kinetic and mechanistic studies of the hydratase-aldolase in the naphthalene pathway (designated NahE) and two hydratase-aldolases in the phenanthrene pathway (PhdG and PhdJ) have been completed. Crystallographic work on two of the enzymes (NahE and PhdJ) provides a rudimentary picture of the mechanism and a platform for future work to identify the structural basis for catalysis and the individual specificities of these hydratase-aldolases.

The Enterococcal Cytolysin Synthetase Coevolves with Substrate for Stereoselective Lanthionine Synthesis.

Stereochemical control is critical in natural product biosynthesis. For ribosomally synthesized and post-translationally modified peptides (RiPPs), the mechanism(s) by which stereoselectivity is achieved is still poorly understood. In this work, we focused on the stereoselective lanthionine synthesis in lanthipeptides, a major class of RiPPs formed by the addition of Cys residues to dehydroalanine (Dha) or dehydrobutyrine (Dhb). Nonenzymatic cyclization of the small subunit of a virulence lanthipeptide, the enterococcal cytolysin, resulted in the native modified peptide as the major product, suggesting that both regioselectivity and stereoselectivity are inherent to the dehydrated peptide sequence. These results support previous computational studies that a Dhx-Dhx-Xxx-Xxx-Cys motif (Dhx = Dha or Dhb; Xxx = any amino acid except Dha, Dhb, and Cys) preferentially cyclizes by attack on the Re face of Dha or Dhb. Characterization of the stereochemistry of the products formed enzymatically with substrate mutants revealed that the lanthionine synthetase actively reinforces Re face attack. These findings support the hypothesis of substrate-controlled selectivity in lanthionine synthesis but also reveal likely coevolution of substrates and lanthionine synthetases to ensure the stereoselective synthesis of lanthipeptides with defined biological activities.

Mechanistic studies on the substrate-tolerant lanthipeptide synthetase ProcM.

Lanthipeptides are a class of post-translationally modified peptide natural products. They contain lanthionine (Lan) and methyllanthionine (MeLan) residues, which generate cross-links and endow the peptides with various biological activities. The mechanism of a highly substrate-tolerant lanthipeptide synthetase, ProcM, was investigated herein. We report a hybrid ligation strategy to prepare a series of substrate analogues designed to address a number of mechanistic questions regarding catalysis by ProcM. The method utilizes expressed protein ligation to generate a C-terminal thioester of the leader peptide of ProcA, the substrate of ProcM. This thioester was ligated with a cysteine derivative that resulted in an alkyne at the C-terminus of the leader peptide. This alkyne in turn was used to conjugate the leader peptides to a variety of synthetic peptides by copper-catalyzed azide–alkyne cycloaddition. Using deuterium-labeled Ser and Thr in the substrate analogues thus prepared, dehydration by ProcM was established to occur from C-to-N-terminus for two different substrates. Cyclization also occurred with a specific order, which depended on the sequence of the substrate peptides. Furthermore, using orthogonal cysteine side-chain protection in the two semisynthetic peptide substrates, we were able to rule out spontaneous non-enzymatic cyclization events to explain the very high substrate tolerance of ProcM. Finally, the enzyme was capable of exchanging protons at the α-carbon of MeLan, suggesting that ring formation could be reversible. These findings are discussed in the context of the mechanism of the substrate-tolerant ProcM, which may aid future efforts in lanthipeptide engineering.

New Bioactive Oxylipins Formed by Non‐Enzymatic Free‐Radical‐Catalyzed Pathways: the Phytoprostanes

In animals and plants, fatty acids with at least three double bonds can be oxidized to prostaglandin-like compounds via enzymatic and non-enzymatic pathways. The most common fatty acid precursor in mammals is arachidonic acid (C20:4) (AA) which can be converted through the cyclooxygenase pathway to a series of prostaglandins (PG). Non-enzymatic cyclization of arachidonate yields a series of isoprostanes (IsoP) which comprises all PG (minor compounds) as well as PG isomers that cannot be formed enzymatically. In contrast, in plants, alpha-linolenic acid (C18:3) (ALA) is the most common substrate for the allene oxide synthase pathway leading to the jasmonate (JA) family of lipid mediators. Non-enzymatic oxidation of linolenate leads to a series of C18-IsoPs termed dinor IsoP or phytoprostanes (PP). PP structurally resemble JA but cannot be formed enzymatically. We will give an overview of the biological activity of the different classes of PP and also discuss their analytical applications and the strategies developed so far for the total synthesis of PP, depending on the synthetic approaches according to the targets and which key steps serve to access the natural products.

Non-enzymatic cyclization of creatine ethyl ester to creatinine

Creatine ethyl ester was incubated at 37 °C in both water and phosphate-buffered saline and the diagnostic methylene resonances in the 1H NMR spectrum were used to identify the resultant products. It was found that mild aqueous conditions result in the cyclization of creatine ethyl ester to provide inactive creatinine as the exclusive product, and this transformation becomes nearly instantaneous as the pH approaches 7.4. This study demonstrates that mild non-enzymatic conditions are sufficient for the cyclization of creatine ethyl ester into creatinine, and together with previous results obtained under enzymatic conditions suggests that there are no physiological conditions that would result in the production of creatine. It is concluded that creatine ethyl ester is a pronutrient for creatinine rather than creatine under all physiological conditions encountered during transit through the various tissues, thus no ergogenic effect is to be expected from supplementation.

Unique Properties of Plasmodium falciparum Porphobilinogen Deaminase

The hybrid pathway for heme biosynthesis in the malarial parasite proposes the involvement of parasite genome-coded enzymes of the pathway localized in different compartments such as apicoplast, mitochondria, and cytosol. However, knowledge on the functionality and localization of many of these enzymes is not available. In this study, we demonstrate that porphobilinogen deaminase encoded by the Plasmodium falciparum genome (PfPBGD) has several unique biochemical properties. Studies carried out with PfPBGD partially purified from parasite membrane fraction, as well as recombinant PfPBGD lacking N-terminal 64 amino acids expressed and purified from Escherichia coli cells (DeltaPfPBGD), indicate that both the proteins are catalytically active. Surprisingly, PfPBGD catalyzes the conversion of porphobilinogen to uroporphyrinogen III (UROGEN III), indicating that it also possesses uroporphyrinogen III synthase (UROS) activity, catalyzing the next step. This obviates the necessity to have a separate gene for UROS that has not been so far annotated in the parasite genome. Interestingly, DeltaPfP-BGD gives rise to UROGEN III even after heat treatment, although UROS from other sources is known to be heat-sensitive. Based on the analysis of active site residues, a DeltaPfPBGDL116K mutant enzyme was created and the specific activity of this recombinant mutant enzyme is 5-fold higher than DeltaPfPBGD. More interestingly, DeltaPfPBGDL116K catalyzes the formation of uroporphyrinogen I (UROGEN I) in addition to UROGEN III, indicating that with increased PBGD activity the UROS activity of PBGD may perhaps become rate-limiting, thus leading to non-enzymatic cyclization of preuroporphyrinogen to UROGEN I. PfPBGD is localized to the apicoplast and is catalytically very inefficient compared with the host red cell enzyme.

Metabolism of 1-(3-[3-(4-cyanobenzyl)-3H-imidazol-4-yl]-propyl)-3-(6-methoxypyridin-3-yl)-1-(2-trifluoromethylbenzyl)thiourea (YH3945), a novel anti-cancer drug, in rats using the 14C-labeled compound.

The metabolism of a novel anti-cancer agent, 1-(3-[3-(4-cyanobenzyl)-3H-imidazol-4-yl]-propyl)-3-(6-methoxypyridin-3-yl)-1-(2-trifluoromethylbenzyl)thiourea (YH3945), was investigated in rats. Bile, plasma, feces, and urine were collected and analyzed by a high-performance liquid chromatography (HPLC) system equipped with ultraviolet (UV), mass spectrometric, and radioactivity detectors. After intravenous dosing, mean radiocarbon recovery was 74.4 +/- 1.3% with 62.4 +/- 1.2% in the feces and 12.0 +/- 0.5% in the urine. Biliary excretion of the radioactivity for the first 24 h period was approximately 32%, suggesting that YH3945 is cleared by hepatobiliary excretion. YH3945 was extensively metabolized to 21 different metabolites including glucuronide conjugates, and structures of the metabolites were elucidated based on MS(n) and NMR spectral analyses. The major metabolic pathways in the rat were identified as O-demethylation of methoxypyridine, N-debenzylation of imidazole, and hydroxylation. Cyclic metabolites were also identified; concomitant demethylation in the methoxypyridine moiety and hydroxylation at the C16 position might destroy the chemical stability of the compound and subsequently lead to non-enzymatic cyclization. Cyclic metabolites were characteristic of YH3945, and a non-enzymatic reaction mechanism for the formation of cyclic metabolites was postulated.

The Dynamic Structure of Jadomycin B and the Amino Acid Incorporation Step of Its Biosynthesis

Jadomycin B, an antifungal antibiotic with a unique 8H-benz[b]oxazolo[3,2-f]phenanthridine pentacyclic skeleton produced by the bacterium Streptomyces venezuelae ISP 5230, exists in a dynamic equilibrium of two diastereomers differing in the configuration of C-3a. Several novel jadomycins with various amino acid-derived 1-side chains could be generated, by replacing isoleucine in the production medium of S. venezuelae with other amino acids. These two findings led to the conclusion that a nonenzymatic reaction with the amino acid followed by a likewise nonenzymatic cyclization cascade are crucial for its late biosynthesis.

A Model for the Nonenzymatic BCD Cyclization of Squalene This research was supported by FWO-Vlaanderen. We thank Dr. Davide Proserpio (Università di Milano) and Dr. Annalisa Guerri (Università di Firenze) for the allowance to use the CCD diffractometers.

A Model for the Nonenzymatic BCD Cyclization of Squalene This research was supported by FWO-Vlaanderen. We thank Dr. Davide Proserpio (Università di Milano) and Dr. Annalisa Guerri (Università di Firenze) for the allowance to use the CCD diffractometers.

On the specificity of allene oxide cyclase.

Jasmonic acid is a carbocyclic fatty acid that is biosynthesized from alpha-linolenic acid in several steps. The formation of the ring structure of jasmonic acid is catalyzed by the enzyme allene oxide cyclase (EC 5.3.99.6) and involves the cyclization of an unstable allene oxide into the cyclopentenone 12-oxo-10,15(Z)-phytodienoic acid. In this study, a number of allene oxides were generated, and their enzymatic and nonenzymatic cyclization into cyclopentenones was investigated. Nonenzymatic cyclization was observed with allene oxides having one pair of conjugated double bonds and an additional isolated double bond in the beta,gamma position relative to the epoxide group, i.e., the partial structure 4,5-epoxy-1,3,7-octatriene. Enzymatic cyclization took place provided that this structural element was inserted in the fatty acid chain with its epoxide group in the n-6,7 position and the isolated double bond in the n-3 position. A number of oxygenated fatty acids having structural features in common with the natural allene oxides were tested as inhibitors of allene oxide cyclase. Fatty acids having an allene oxide structure in the n-6,7 position but lacking the double bond in the n-3 position, as well as fatty acids having a saturated epoxide group in the n-6,7 position, served as competitive inhibitors of the enzyme. Data on the substrate specificity of allene oxide synthase (EC 4.2.1.92) from corn seeds indicated that fatty acid hydroperoxides with a double bond at n-3 and with the hydroperoxide function at n-6 exhibit the highest affinity but the slowest reaction velocity.

Development of a Continuous, Fluorometric Coupled Enzyme Assay for Thyrotropin-Releasing Hormone-Degrading Ectoenzyme

Thyrotropin-releasing hormone degrading-ectoenzyme (TRH-DE) (EC 3.4.19.6), removes the N-terminal pyroglutamyl residue of thyrotropin-releasing hormone (TRH). Discontinuous assays have been used to measure TRH-DE activity; however, a continuous assay is needed to make reliable measurements of initial rates and facilitate kinetic studies. Presented is a continuous, coupled enzyme assay for TRH-DE in which TRH-DE hydrolyzed the substrate, pyroglutamyl-histidyl-prolylamido-4-methyl coumarin (TRHMCA), to give His-ProMCA, which was then cleaved by dipeptidyl peptidase IV (EC 3.4.14.5) to give 7-amino-4-methyl coumarin (MCA). Reaction progress was monitored continuously by measuring the increase in MCA fluorescence. This assay should be especially useful for rapid screening of potential TRH-DE inhibitors. A previously reported discontinuous assay, where nonenzymatic cyclization at 80°C was used to liberate MCA from His-ProMCA, was found to underestimate the amount of product formed. A modified procedure that avoids this is presented. Initial rates and kinetic parameters for TRHMCA hydrolysis by TRH-DE determined using this modified assay correspond with those determined by the continuous assay. Discontinuous and continuous assays gave Km values for TRHMCA of 3.4 ± 0.7 μM (n = 5) and 3.8 ± 0.5 μM (n = 5), respectively. Ki values determined by the discontinuous assay for TRH and TRH-OH were 35 ± 4 μM (n = 3) and 311 ± 31 μM (n = 5), respectively.

PEGylation prevents the N-terminal degradation of megakaryocyte growth and development factor.

Determine the effect of PEGylation on in-vitro degradation for recombinant human Megakaryocyte Growth and Development Factor (rHuMGDF) in the neutral pH range. Degradation products were characterized by cation-exchange HPLC, N-terminal sequencing and mass spectrometry. The main route of degradation was through non-enzymatic cyclization of the first two amino acids and subsequent cleavage to form a diketopiperazine and des(Ser, Pro)rHuMGDE This reaction was prevented by alkylation of the N-terminus by polyethylene glycol (PEG). PEGylation of proteins is commonly performed to achieve increased in-vivo circulation half-lives. For rHuMGDF, an additional advantage of PEGylation was enhanced in-vitro shelf-life stability.

The Development of Two Fluorimetric Assays for the Determination of Pyroglutamyl Aminopeptidase Type-II Activity

Two fluorimetric assays for the determination of pyroglutamyl aminopeptidase type-II activity have been developed. The assays are based on hydrolysis of the quenched-fluorimetric substrate <Glu-His-Pro-7-amino-4-methylcoumarin. Following the removal of the N-terminal <Glu by pyroglutamyl aminopeptidase type-II, liberation of 7-amino-4-methylcoumarin from the metabolite His-Pro-7-amino-4-methylcoumarin is catalyzed by one of two methods: (i) the addition of partially purified bovine serum dipeptidyl aminopeptidase type-IV or (ii) by incubating the reaction mixture for up to 2 h at 80°C, thus promoting the nonenzymatic cyclization of His-Pro-7-amino-4-methylcoumarin to cyclo His-Pro and free 7-amino-4-methylcoumarin. Pyroglutamyl aminopeptidase type-II from bovine brain is used to establish appropriate assay conditions. These fluorimetric assays offer expeditious alternatives to the existing radiolabeled thyrotropin-releasing hormone assays for the determination of PAPII activity.

Diketopiperazine formation and N‐terminal degradation in recombinant human growth hormone

A new degradation process has been identified that occurs in recombinant DNA-derived human growth hormone. Non-enzymatic cyclization of the first two amino acids from the N-terminus and subsequent cleavage results in the formation of a diketopiperazine and a truncated variant of rhGH. The truncated protein was separated using hydrophobic interaction chromatography and identified as desPhe1Pro2-rhGH using N-terminal sequence analysis, tryptic mapping, and mass spectrometry.