Evaluation of aminolysis of anilinothiazolinones to phenylthiocarbamoyl amino acid methyl amides as an alternative conversion method in protein sequencing.

The aminolysis of products of sequential degradation of proteins and peptides by methylamine is an alternative method of conversion of the unstable 5-alkyl-2-anilino-4-thiazolinones into the stable methyl amides of N alpha-phenylthiocarbamoyl amino acids. The volatility of methylamine permits use in the gas phase during both manual and automatic sequential degradation. Two procedures were studied: (mode A) aminolysis by methylamine in the sequencer reaction chamber after liberation of the thiazolinones by trifluoroacetic acid and (mode B) aminolysis by methylamine vapors passed through a 1-chlorobutane solution of thiazolinones in the conversion flask of the sequencer. The sequencing program was modified for both procedures by making use of the standard sequencer functions. The yields of aminolysis in the conversion flask (mode B) are comparable to those obtained by standard conversion in 25% trifluoracetic acid and the procedure does not affect the repetitive yield. Aminolysis on the glass filter (mode A) requires a major modification of the degradation process, yet gives higher yields of the degraded amino acid derivatives. A disadvantage of both procedures, especially of mode A, is the presence of N-methyl-N'-phenylthiourea in the methyl amide samples. We have not been able to achieve the expected improvement of the yields of degraded hydroxy amino acids. Therefore the replacement of acid conversion of anilinothiazolinones to phenylthiohydantiones by aminolysis for routine degradation cannot be recommended. High yields of methyl amides make aminolysis a promising candidate for the incorporation of fluorescent or other labels in the products of sequencing degradation.     

Microsequence analysis of peptides and proteins. IX. An improved, compact, automated instrument

We describe the construction of an improved, compact protein sequencer with a vertical flow path and continuous flow reactor (CFR). Unique features include a hexagonal valve for six fluid inputs to the CFR, which connects vertically to a transfer valve that allows sample, reagent, and solvent input to a conversion flask (CF). The simplified CF contains only two inputs at the top, one for sample, reagent, and solvent input, and the other a vent. The CF drains from the bottom, connecting to a switching valve which allows either delivery to waste or to an on-line HPLC for the analysis of phenylthiohydantoin amino acid derivatives. Approximately 90% of the sample is analyzed by use of a sonic flow detector. The overall vertical flow path of the sequencer is about 16 cm. The size of the instrument (25 w x 38 x 44 d cm) is smaller than that of commercially available sequencers or HPLC systems. The performance of the instrument includes reduced background peaks and high-sensitivity sequence analysis at the 5-10 pmol level. The simplified sequencer is more economical and portable than conventional sequencers.     

Posttranslational modification of tubulin by palmitoylation: II. Identification of sites of palmitoylation.

As shown in the companion article, tubulin is posttranslationally modified in vivo by palmitoylation. Our goal in this study was to identify the palmitoylation sites by protein structure analysis. To obtain quantities of palmitoylated tubulin required for this analysis, a cell-free system for enzymatic [3H]palmitoylation was developed and characterized in our companion article. We then developed a methodology to examine directly the palmitoylation of all 451 amino acids of alpha-tubulin. 3H-labeled palmitoylated alpha-tubulin was cleaved with cyanogen bromide (CNBr). The CNBr digest was resolved according to peptide size by gel filtration on Sephadex LH60 in formic acid:ethanol. The position of 3H-labeled palmitoylated amino acids in peptides could not be identified by analysis of the Edman degradation sequencer product because the palmitoylated sequencer products were lost during the final derivatization step to phenylthiohydantoin derivatives. Modification of the gas/liquid-phase sequencer to deliver the intermediate anilinothiozolinone derivative, rather than the phenylthiohydantoin derivative, identified the cycle containing the 3H-labeled palmitoylated residue. Therefore, structure analysis of peptides obtained from gel filtration necessitated dual sequencer runs of radioactive peptides, one for sequence analysis and one to identify 3H-labeled palmitoylated amino acids. Further cleavage of the CNBr peptides by trypsin and Lys-C protease, followed by gel filtration on Sephadex LH60 and dual sequencer runs, positioned the 3H-labeled palmitoylated amino acid residues in peptides. Integration of all the available structural information led to the assignment of the palmitoyl moiety to specific residues in alpha-tubulin. The palmitoylated residues in alpha-tubulin were confined to cysteine residues only. The major site for palmitoylation was cysteine residue 376.     

Consolidated conversion of protein waste into biofuels and ammonia using Bacillus subtilis

The non-recyclable use of nitrogen fertilizers in microbial production of fuels and chemicals remains environmentally detrimental. Conversion of protein wastes into biofuels and ammonia by engineering nitrogen flux in Escherichia coli has been demonstrated as a method to reclaim reduced-nitrogen and curb its environmental deposition. However, protein biomass requires a proteolysis process before it can be taken up and converted by any microbe. Here, we metabolically engineered Bacillus subtilis to hydrolyze polypeptides through its secreted proteases and to convert amino acids into advanced biofuels and ammonia fertilizer. Redirection of B. subtilis metabolism for amino-acid conversion required inactivation of the branched-chain amino-acid (BCAA) global regulator CodY. Additionally, the lipoamide acyltransferase (bkdB) was deleted to prevent conversion of branched-chain 2-keto acids into their acyl-CoA derivatives. With these deletions and heterologous expression of a keto-acid decarboxylase and an alcohol dehydrogenase, the final strain produced biofuels and ammonia from an amino-acid media with 18.9% and 46.6% of the maximum theoretical yield. The process was also demonstrated on several waste proteins. The results demonstrate the feasibility of direct microbial conversion of polypeptides into sustainable products.     

Sensitization of Edman amino acid derivatives using the fluorescent reagent, 4-aminofluorescein

The ability to analyze amino acid derivatives at the femtomole level is one of the most interesting challenges in the field of protein microsequencing. 2-Anilino-5-thiazolinone amino acids, obtained by Edman degradation, were quantitatively derivatized with fluorescent primary amines. The most fluorescent reagent tested was 4-aminofluorescein. The amino acid derivatives sensitized with this reagent were separated using reversed-phase high-performance liquid chromatography and identified at the 100 attomole level. Incorporation of this method into the operation of a conventional automated sequencer is also described.     

A rapid solid-phase protein microsequencer.

A solid-phase protein microsequencer is described that has been designed to determine protein sequences with subnanomolar quantities of protein. Its utility has been demonstrated by the determination of many sequences in subunits of mitochondrial F1-ATPase, in a protein isolated from mouse gap junctions and in the mitochondrial phosphate-transporter protein. It has a number of advantages over liquid- and gas-phase sequencers. Firstly, the degradation cycle takes 24 min, more than twice as fast as any other sequencer. This helps to reduce exposure of proteins to inimical reagents and increases throughput of samples. Secondly, polar amino acids such as phosphoserine, and polar derivatives formed by active-site photoaffinity labelling with 8-azido-ATP, are recovered quantitatively from the reaction column and can be positively identified. In other types of sequencer these polar derivatives, being somewhat insoluble in butyl chloride, tend to remain in the reaction chamber of the instrument and so are more difficult to identify. The solid-phase protein sequencer is also more suited than the liquid-phase instrument for analysis of proteolipids from membranes. These hydrophobic proteins tend to dissolve in organic solvents during washing steps in the liquid-phase instrument and are lost. Covalent attachment as used in the solid-phase instrument solves this problem.     

Sequence analysis of phosphoserine-containing peptides. Modification for picomolar sensitivity

Sequencing of phosphoserine-containing peptides yields normally no identifiable PTH-derivatives at those positions where phosphoserine is located. Here a new method is described which allows identification of the position of phosphoserine by chemical modification just before sequence analysis. In a one-step microbatch reaction, phosphoserine present in the intact peptide can be transformed quantitatively into stable derivatives such as beta-methylaminoalanine (MAA), S-ethanolcysteine or S-ethylcysteine. These derivatives are detectable during microsequencing with less than 100 pmol peptide using an Applied Biosystems gas-phase sequencer equipped with an on-line PTH amino acid analyzer.     

The amino acid sequence of human sperm protamine P1

Human sperm protamines have been extracted from spermatozoa pooled from several donors, converted to their S-pyridylethylated derivatives and resolved into two major components, P1 and PI, by Bio-Rex 70 chromatography. Protamine P1 was further purified by Bio-Gel P-10 chromatography and sequenced directly on a gas phase protein sequencer for 43 residues. To complete the sequence, P1 was cleaved at methionine 36 and the C-terminal tetradecapeptide was purified by h.p.i.c , and sequenced completely. The 50 residue sequence is: 10 20 30 40 ARYRC CRSQS RSRYY RQRQR SRRRR RRSCQ TRRRA MRCCR 50 PRYRP RCRRH. This sequence has a calculated molecular weight of 6674 and is homologous with four other published mammalian protamine sequences.     

Determination of the metabolic origins of the sulfur and 3'-nitrogen atoms in biotin of Escherichia coli by mass spectrometry

Two steps in the biosynthesis of biotin in Escherichia coli, incorporation of the nitrogen atom of methionine into 7-keto-8-aminopelargonic acid and of the sulfur atom into dethiobiotin, were examined. Sulfur and nitrogen metabolism were monitored by gas chromatography-mass spectrometry of volatile derivatives of internal (protein-bound) amino acids and excreted biotin. We were able to show that internal cysteine and excreted biotin were labeled to the same extent with 34S from either of two exogenous sulfur sources, 34SO4(2)-or L-[sulfane-34S]thiocystine. Internal methionine was eliminated from consideration, while cysteine, or possibly a closely related intermediate, was implicated as providing the sulfur atom for biotin biosynthesis. Also, in experiments designed to follow the metabolism of the nitrogen atom of methionine, it was found that biotin excreted into the culture medium by this organism grown with 95 atom % [15N]methionine contained greater than 70 atom % excess 15N in one of the nitrogens over that obtained from cultures grown with methionine of natural abundance 15N. These results provide evidence for the direct transfer of the methionine nitrogen as the role of S-adenosylmethionine in the conversion of 7-keto-8-aminopelargonic acid to 7,8-diaminopelargonic acid.     

A novel pathway for sequential transformation of 7-dehydrocholesterol and expression of the P450scc system in mammalian skin.

Following up on our previous findings that the skin possesses steroidogenic activity from progesterone, we now show widespread cutaneous expression of the full cytochrome P450 side-chain cleavage (P450scc) system required for the intracellular catalytic production of pregnenolone, i.e. the genes and proteins for P450scc enzyme, adrenodoxin, adrenodoxin reductase and MLN64. Functionality of the system was confirmed in mitochondria from skin cells. Moreover, purified mammalian P450scc enzyme and, most importantly, mitochondria isolated from placenta and adrenals produced robust transformation of 7-dehydrocholesterol (7-DHC; precursor to cholesterol and vitamin D3) to 7-dehydropregnenolone (7-DHP). Product identity was confirmed by comparison with the chemically synthesized standard and chromatographic, MS and NMR analyses. Reaction kinetics for the conversion of 7-DHC into 7-DHP were similar to those for cholesterol conversion into pregnenolone. Thus, 7-DHC can form 7-DHP through P450scc side-chain cleavage, which may serve as a substrate for further conversions into hydroxy derivatives through existing steroidogenic enzymes. In the skin, 5,7-steroidal dienes (7-DHP and its hydroxy derivatives), whether synthesized locally or delivered by the circulation, may undergo UVB-induced intramolecular rearrangements to vitamin D3-like derivatives. This novel pathway has the potential to generate a variety of molecules depending on local steroidogenic activity and access to UVB.     

The metabolism of niacytin in the rat. Trigonelline as a major metabolite of niacytin in the urine

1. To investigate the fate of orally administered niacytin, urine and faeces of rats given single niacytin doses were examined for nicotinic acid derivatives methylated on the pyridine nitrogen atom, determined as trigonelline. 2. Methods were devised for the extraction of trigonelline from urine and faeces and for its differentiation from N'-methylnicotinamide. 3. A prolonged elevation of the excretion of trigonelline in the urine of rats dosed with niacytin was detected colorimetrically, in contrast with the urinary excretion in control groups given free nicotinic acid or hydrolysed niacytin. The total conversion of the nicotinoyl moiety of niacytin into trigonelline was 30-40%. 4. The identity of this metabolite as trigonelline was established by t.l.c., by its u.v. spectrum and by g.l.c. after conversion into methyl nicotinate. 5. The excretion of Ehrlich-positive substances was also increased in urine after administration of niacytin, the increase being approximately parallel to the trigonelline excretion. 6. No increase in the excretion of trigonelline in faeces was found after administration of niacytin. 7. These results suggest a metabolic path-way for niacytin in the rat involving methylation of the pyridine nitrogen without prior release of free nicotinic acid. This hypothesis explains the absence of biological activity of niacytin. An endogenous source of urinary trigonelline was also demonstrated.     

Isocratic separation of PTH-amino acids at picomole level by reverse-phase HPLC in the presence of sodium dodecylsulfate

The phenylthiohydantoin (PTH) derivatives of protein amino acids have been separated by reverse-phase high performance liquid chromatography (HPLC) on a fully end-capped C18 column using an isocratic solvent system. The developing solvent was 0.01 M sodium acetate buffer (pH 4.5) containing 39.5% acetonitrile and 0.02% sodium dodecylsulfate (SDS). With an automated liquid chromatography equipped with a dual-channel detector, operating at 254 and 313 nm, the present isocratic separation system was quite useful for routine microanalysis of PTH-amino acids released with a "gas-phase" sequencer. The time for one run was approximately 23 min and the limit of analysis approximately 2.5 pmol of a PTH-amino acid.     

Selectively blocked derivatives of muco-inositol and their conversion into derivatives of epi- and cis-inositol.

Benzylation, and then hydrolysis, of 1, 2:4, 5-di-O-isopropylidene-muco-inositol (1) gave 3, 6-di-O-benzyl-muco-inositol (3). This was converted into a series of derivatives, including the 1, 5-di-O-benzoyl-3, 6-di-O-benzyl-2, 4-di-p-toluenesulfonate 7. The resistance to displacement of the sulfonate groups in 7 prevented conversion of 7 into an intermediate for the synthesis of aminoglycoside antibiotics. Monobenzylation of 1, followed by an oxidation-reduction cycle, yielded 6-O-benzyl-1, 2:4, 5-di-O-isopropylidene-epi-inositol (10). From this was synthesized a series of epi-inositol derivatives, analogous to the muco series but less complete. For derivatives of 1, 2:5, 6-di-O-isopropylidene-epi- and muco-inositol, the p. m. r. data indicate modified skew conformations. The reaction of the 3, 6-di-p-bromobenzenesulfonate (17) of 1 with anhydrous hydrazine proceeded in part by S-O cleavage to regenerate 1, and in part by displacement of both sulfonate groups by the same nitrogen atom. The resulting, novel 1, 4-epimino-cis-inositol was converted into further derivatives.     

Determination of the site of tyrosine phosphorylation at the low picomole level by automated solid-phase sequence analysis.

A method for the determination of the sites of tyrosine phosphorylation in proteins and peptides at the low picomole level for "cold" phosphopeptides and at the subpicomole level for 32P-labeled phosphopeptides is presented. The procedure is based on solid-phase sequence analysis of phosphopeptides immobilized on carrier discs and the "on-line" detection by reverse-phase high-performance liquid chromatography of the phenylthiohydantoin derivative of phosphotyrosine. The procedure is sensitive and automated and allows the identification of phosphotyrosine derivatives in the same operation as the detection of the derivatives of the other common amino acids. Essentially quantitative extraction of the phosphotyrosine derivatives from the sequencer makes this method ideally suited for the quantitative assessment of protein-tyrosine kinase and protein phosphatase activities and for the determination of their respective recognition sequences.     

Application of metagenomic techniques in mining enzymes from microbial communities for biofuel synthesis

Feedstock for biofuel synthesis is transitioning to lignocelluosic biomass to address criticism over competition between first generation biofuels and food production. As microbial catalysis is increasingly applied for the conversion of biomass to biofuels, increased import has been placed on the development of novel enzymes. With revolutionary advances in sequencer technology and metagenomic sequencing, mining enzymes from microbial communities for biofuel synthesis is becoming more and more practical. The present article highlights the latest research progress on the special characteristics of metagenomic sequencing, which has been a powerful tool for new enzyme discovery and gene functional analysis in the biomass energy field. Critical enzymes recently developed for the pretreatment and conversion of lignocellulosic materials are evaluated with respect to their activity and stability, with additional explorations into xylanase, laccase, amylase, chitinase, and lipolytic biocatalysts for other biomass feedstocks.     

Bioconversion of glyceryl trinitrate into mononitrates by Geotrichum candidum

Glyceryl trinitrate (GTN) 2 mM was quantitatively converted into its 1 and 2 mononitrate derivatives by Geotrichum candidum, with consumption of the nitrite ions produced. The conversion proceeded at a rate independent of the addition of either organic carbon or organic nitrogen sources. Eight batches of nitrate ester, which were added every 24 hours, were successfully converted as far as during the bioconversion process GTN concentration did not exceed 2 mM. When those limiting conditions were not observed, dramatic toxicity of GTN was noticed.     

The N-terminal amino acid sequence of plastocyanin from Stellaria media L. An exercise to establish criteria for the identification of residues from a sequenator.

The N-terminal amino acid sequence of plastocyanin from Stellaria media L. (chickweed) has been determined by means of an automatic sequencer. Amino acid derivatives obtained during the degradation were identified by a combination of t.l.c., g.l.c. and regeneration of the parent amino acid. The analytical results are presented for each sample, and the criteria used to establish the amino acid sequence are discussed.     

Antioxidant activity of 4-flavonil-1,4-dihydropyridine derivatives

The effect of 4-flavonil-1,4-dihydropyridine derivatives on a chemical system involving a superoxide radical anion was tested using the chemiluminescence and spectrophotometry methods. All tested compounds enhanced the light emission from the system. The obtained results indicated that the tested derivatives may catalyze the conversion of superoxide radicals, thus showing superoxide dismutase activity.     

Chemistry and in vitro mutagenicity of 2-aminofluorene derivatives

A study on the relationship between mutagenic activity and chemical reactivity of a series of 2-fluorenylamino and hydroxylamino derivatives has been carried out by assaying their ability to revert the Salmonella typhimurium strain TA98. The mutagenic potency of the fluorenamides increased with increasing availability of the amidic hydrogen for abstraction and tertiary amides were quite inactive. N-Hydroxy and N-acyloxy derivatives were directly mutagenic and increased their mutagenic activity after metabolic conversion by liver S9. N-Hydroxy-2-benzoylaminofluorene, inactive without S9, after activation was the most mutagenic. Of a pair of N-acyloxy-derivatives, N-benzoyloxy-2-acetylaminofluorene, which undergoes rearrangement of the benzoyloxy group from nitrogen to ring carbons even at room temperature, was less potent than N-acetyloxy-2-acetylamino-fluorene whose rearrangement occurs at higher temperatures. Corresponding C-1 and C-3 benzoyloxy and acetyloxy derivatives were found ineffective in this assay in agreement with previous reports on the hydroxy series. N-Chloro-2-amino-(or acetylamino)fluorene were found more active than the corresponding N-hydroxy analogs in the presence of S9, thus suggesting an alternate pathway for activation, likely a direct conversion to electrophilic species. Furthermore, in contrast with inactivity of ring hydroxy and acyloxy derivatives, 3-chloro-2-acetylaminofluorene retained mutagenic activity. Finally 2,2'-azoxyfluorene, the ultimated oxidation product of N-hydroxyaminofluorene, tested in vitro and in vivo experiments, was found completely inactive.     

Protein-based biorefining: metabolic engineering for production of chemicals and fuel with regeneration of nitrogen fertilizers

Threats to stable oil supplies and concerns over environmental emissions have pushed for renewable biofuel developments to minimize dependence on fossil resources. Recent biofuel progress has moved towards fossil resource-independent carbon cycles, but environmental issues regarding use of nitrogen fertilizers have not been addressed on a global scale. The recently demonstrated conversion of waste protein biomass into advanced biofuels and renewable chemicals, while recycling nitrogen fertilizers, offers a glimpse of the efforts needed to balance the nitrogen cycle at scale. In general, the catabolism of protein into biofuels is challenging because of physiological regulation and thermodynamic limitations. This conversion became possible with metabolic engineering around ammonia assimilation, intracellular nitrogen flux, and quorum sensing. This review highlights the metabolic engineering solutions in transforming those cellular processes into driving forces for the high yield of chemical products from protein.