Automation of RNA-based biomarker extraction from dried blood spots for the detection of blood doping.

Aim: Transcriptomic biomarkers originating from reticulocytes measured in dried blood spots (DBSs) may be reliable indicators of blood doping. Methods/results: Here, we examined changes in the expression levels of the erythropoiesis-related ALAS2, CA1 and SLC4A1 genes in DBS samples from elite athletes and volunteers of clinical study with recombinant erythropoietin dose. Conclusion: By comparing the mean intraday coefficients of variation for ALAS2L, ALASLC, CA1 and SLC4A1 between manual and automated RNA extractions, an average improvement was observed, whereas the assessment of interday variability provided comparable results for both manual and automated approaches. Our results confirmed that RNA biomarkers on DBS support are efficient to detect blood doping.

Purification and functional characterization of thermostable 5-aminolevulinic acid synthases.

OBJECTIVES: As 5-aminolevulinic acid synthase (ALAS), the key enzyme for 5-aminolevulinic acid (ALA) synthesis, is unstable, we have sought to find thermostable ALASs from thermophilic organisms. RESULTS: Three ALASs from thermophiles Geobacillus thermoglucosidasius (GT-ALAS), Laceyella sacchari (LS-ALAS) and Pseudomonas alcaliphila (PA-ALAS) were purified and characterized. All enzymes were more stable than two previously studied ALASs from Rhodopseudomonas palustris and Rhodobacter sphaeroides. There was almost no activity change after 60 h at 37 °C for the three thermostable enzymes. This contrasts with the other two enzymes which lost over 90 % activities in just 1 h. Furthermore, the specific activity of LS-ALAS (7.8 U mg(-1)) was also higher than any previously studied ALASs. CONCLUSIONS: Thermostable ALASs were found in thermophilic organisms and this paves the way for developing cell free processes for enzymatic production of ALA from bulk chemicals succinate and glycine.

High-level soluble expression of the hemA gene from Rhodobacter capsulatus and comparative study of its enzymatic properties.

The Rhodobacter capsulatus hemA gene, which encodes 5-aminolevulinic acid synthase (ALAS), was expressed in Escherichia coli Rosetta (DE3) and the enzymatic properties of the purified recombinant ALAS (RC-ALAS) were studied. Compared with ALASs encoded by hemA genes from Agrobacterium radiobacter (AR-ALAS) and Rhodobacter sphaeroides (RS-ALAS), the specific activity of RC-ALAS reached 198.2 U/mg, which was about 31.2% and 69.5% higher than those of AR-ALAS (151.1 U/mg) and RS-ALAS (116.9 U/mg), respectively. The optimum pH values and temperatures of the three above mentioned enzymes were all pH 7.5 and 37 °C, respectively. Moreover, RC-ALAS was more sensitive to pH, while the other two were sensitive to temperature. The effects of metals, ethylene diamine tetraacetic acid (EDTA), and sodium dodecyl sulfate (SDS) on the three ALASs were also investigated. The results indicate that they had the same effects on the activities of the three ALASs. SDS and metal ions such as Co(2+), Zn(2+), and Cu(2+) strongly inhibited the activities of the ALASs, while Mn(2+) exerted slight inhibition, and K(+), Ca(2+), Ba(2+), Mg(2+), or EDTA had no significant effect. The specificity constant of succinyl coenzyme A [(kcat/Km)(S-CoA)] of RC-ALAS was 1.4989, which was higher than those of AR-ALAS (0.7456) and RS-ALAS (1.1699), showing its high catalytic efficiency. The fed-batch fermentation was conducted using the recombinant strain containing the R. capsulatus hemA gene, and the yield of 5-aminolevulinic acid (ALA) achieved was 8.8 g/L (67 mmol/L) under the appropriate conditions.

A three enzyme pathway for 2-amino-3-hydroxycyclopent-2-enone formation and incorporation in natural product biosynthesis.

A number of natural products contain a 2-amino-3-hydroxycyclopent-2-enone five membered ring, termed C(5)N, which is condensed via an amide linkage to a variety of polyketide-derived polyenoic acid scaffolds. Bacterial genome mining indicates three tandem ORFs that may be involved in C(5)N formation and subsequent installation in amide linkages. We show that the protein products of three tandem ORFs (ORF33-35) from the ECO-02301 biosynthetic gene cluster in Streptomyces aizunenesis NRRL-B-11277, when purified from Escherichia coli, demonstrate the requisite enzyme activities for C(5)N formation and amide ligation. First, succinyl-CoA and glycine are condensed to generate 5-aminolevulinate (ALA) by a dedicated PLP-dependent ALA synthase (ORF34). Then ALA is converted to ALA-CoA through an ALA-AMP intermediate by an acyl-CoA ligase (ORF35). ALA-CoA is unstable and has a half-life of approximately 10 min under incubation conditions for off-pathway cyclization to 2,5-piperidinedione. The ALA synthase can compete with the nonenzymatic decomposition route and act in a novel second transformation, cyclizing ALA-CoA to C(5)N. C(5)N is then a substrate for the third enzyme, an ATP-dependent amide synthetase (ORF33). Using octatrienoic acid as a mimic of the C(56) polyenoic acid scaffold of ECO-02301, formation of the octatrienyl-C(5)N product was observed. This three enzyme pathway is likely the general route to the C(5)N ring system in other natural products, including the antibiotic moenomycin.

Characterization of 5-aminolevulinate synthase from Agrobacterium radiobacter, screening new inhibitors for 5-aminolevulinate dehydratase from Escherichia coli and their potential use for high 5-aminolevulinate production.

The hemA gene encoding 5-aminolevulinate synthase (ALAS) from Agrobacterium radiobacter zju-0121 showed 92.6% homology with that from A. radiobacter ATCC4718 and contained several rare codons. To enhance the expression of this gene, Escherichia coli Rosetta(DE3), which is a rare codon optimizer strain, was used as the host to construct an efficient recombinant strain. And the encoded protein was over-expressed as fusion protein and was purified by affinity purification on Ni-NTA agarose and by gel filtration chromatography on Sephadex G-25 Medium resin. The recombinant protein was partly characterized, and D-glucose, D-fructose, D-xylose, D-mannose, L-arabinose, D-galactose, lactose, sucrose and maltose were detected to have no distinct inhibition on this recombinant ALAS. Meanwhile, 20mM D-glucose or D-xylose inhibited about 20% activity of ALA dehydratase (ALAD) from Escherichia coli Rosetta(DE3). Combining D-xylose as a new inhibitor for ALAD with D-glucose in fed-batch culture and based on the optimal culture system using Rosetta(DE3)/pET28a-hemA, the yield of ALA achieved was 7.3g/l (56 mM) under the appropriate conditions in the fermenter.

Expression and purification of mammalian 5-aminolevulinate synthase.

We have described a procedure for production and purification of recombinant, mature-length mouse ALAS-2. The fact that E. coli utilizes the C5 path for ALA production means that there is no problem with contamination of the recombinant ALAS-2 by host cell enzyme, such as one may have with a yeast expression system. While the detailed procedure produces enzyme in good yield with relatively common protein purification techniques, future expression systems may be developed to take advantage of the rapid purification achieved by the use of a 6-histidine (His6) aminoterminal tag and metal chelate chromatography. Such approaches in this laboratory with protoporphyrinogen oxidase, coproporphyrinogen oxidase, and uroporphyrinogen decarboxylase have resulted in the production and purification of enzymes whose kinetic and physical parameters are essentially identical to those of proteins lacking the His6 tag.

Aminolevulinate synthase: lysine 313 is not essential for binding the pyridoxal phosphate cofactor but is essential for catalysis.

5-Aminolevulinate synthase is the first enzyme of the heme biosynthetic pathway in animals and some bacteria. Lysine-313 of the mouse erythroid aminolevulinate synthase was recently identified to be linked covalently to the pyridoxal 5'-phosphate cofactor (Ferreira GC, Neame PJ, Dailey HA, 1993, Protein Sci 2:1959-1965). Here we report on the effect of replacement of aminolevulinate synthase lysine-313 by alanine, histidine, and glycine, using site-directed mutagenesis. Mutant enzymes were purified to homogeneity, and the purification yields were similar to those of the wild-type enzyme. Although their absorption spectra indicate that the mutant enzymes bind pyridoxal 5'-phosphate, they bind noncovalently. However, addition of glycine to the mutant enzymes led to the formation of external aldimines. The formation of an external aldimine between the pyridoxal 5'-phosphate cofactor and the glycine substrate is the first step in the mechanism of the aminolevulinate synthase-catalyzed reaction. In contrast, lysine-313 is an essential catalytic residue, because the K313-directed mutant enzymes have no measurable activity. In summary, site-directed mutagenesis of the aminolevulinate synthase active-site lysine-313, to alanine (K313A), histidine (K313H), or glycine (K313G) yields enzymes that bind the pyridoxal 5'-phosphate cofactor and the glycine substrate to produce external aldimines, but which are inactive. This suggests that lysine-313 has a functional role in catalysis.

Purification and structure of rat erythroid-specific delta-aminolevulinate synthase.

The existence of erythroid form delta-aminolevulinate synthase (ALAS-E) was historically a matter of some controversy. To obtain direct evidence for a unique ALAS-E, we have purified ALAS-E to homogeneity for the first time, from rat reticulocyte lysate. The papain digestion method was used at the initial step of the purification to overcome the difficulty which repeatedly hampered earlier attempts to purify ALAS-E. The size of the purified papain-resistant core catalytic domain of ALAS-E was estimated electrophoretically to be 49,000 Da. The pH optimum (7.6) and apparent Km values for the substrates, glycine (6.5 mM) and succinyl-CoA (2 microM), were similar to those of the non-specific form of delta-aminolevulinate synthase (ALAS-N); but, in contrast to ALAS-N, the substrate inhibition by succinyl-CoA was not evident in ALAS-E. We then isolated cDNA and genomic DNA clones encoding rat ALAS-E. By combining the nucleotide sequence information of the cDNA and genomic clones, the rat ALAS-E precursor is predicted to be composed of 587 amino acids with a calculated molecular mass of 64,841 Da. All the peptide sequences determined directly from the purified protein agreed with those predicted from the nucleotide data, demonstrating the existence of ALAS-E. Analysis of the papain-resistant core domain further revealed that it overlaps with the evolutionally conserved segment that has been noticed by sequence alignment analysis of ALA synthases from various species.

Expression of mammalian 5-aminolevulinate synthase in Escherichia coli. Overproduction, purification, and characterization.

5-Aminolevulinate synthase catalyzes the first step of the heme biosynthetic pathway in nonplant higher eukaryotes. A cDNA encoding for the mouse erythroid 5-aminolevulinate synthase (Schoenhaut, D. S., and Curtis, P.J. (1986) Gene (Amst.) 48, 55-63) has been expressed in Escherichia coli, using the alkaline phosphatase promoter, to a level of 50-60% of the total bacterial protein. Aminolevulinate synthase was overexpressed in an active form and, therefore, was able to rescue hemA mutants, which are unable to grow in the absence of 5-aminolevulinate. A simple purification from the aminolevulinate synthase-overproducing bacterial strain yielded approximately 50 mg of protein, in a high state of purity, per liter of bacterial culture. Moreover, the expressed aminolevulinate synthase could be easily concentrated up to 6-8 mg/ml. Significantly, recombinant aminolevulinate synthase retained physical and catalytic properties identical to those of natural sources. These include the dimeric structure, subunit molecular mass, and pyridoxal 5'-phosphate as an essential cofactor. Removal of the pyridoxal 5'-phosphate led to complete loss of activity. However, the apoenzyme could be readily reconstituted by incubation with 20 microM 5'-pyridoxal phosphate. The Km values are 51 mM for glycine and 55 microM for succinyl-CoA, in the same range of the Km values determined for the nonrecombinant enzyme. This report describes the overexpression of a mammalian 5-aminolevulinate synthase in E. coli and its purification from an overproducing strain. The ready availability of the pure, cloned, sequenced erythroid 5-aminolevulinate synthase makes it possible now for questions pertinent to the enzyme's structure, mechanism, and regulation to be addressed.

Trout (Salmo gairdneri R.) liver 5-aminolevulinate synthetase: subcellular distribution and properties.

The subcellular distribution of trout liver 5-aminolevulinate synthetase has been studied. A cytosolic form of the enzyme has been found. Its activity was a 30% of the mitochondrial enzyme. The cytosolic form has a molecular weight of 110,000, larger than the mitochondrial enzyme (70,000). The two enzyme forms showed a pH optimum of 7.5. The kinetic characteristic of both forms suggest that the cytosolic form is a precursor of the mitochondrial ALA-synthetase.

Isolation and properties of 5-aminolevulinate synthase from the yeast Saccharomyces cerevisiae.

5-Aminolevulinate synthase from yeast mitochondria has been purified to homogeneity for the first time. By using affinity chromatography on agarose-hexane-CoA, gel filtration and DEAE-Sepharose chromatography, the enzyme was purified about 7000-fold with an overall yield of 40%. The specific activity of the final preparation was 39000 nmol of 5-aminolevulinate h-1 mg-1 of protein at 30 degrees C. As judged by gel filtration, polyacrylamide gradient gel and sodium dodecyl sulfate/polyacrylamide gel electrophoresis, the enzyme appeared to be composed of two identical subunits of a relative molecular mass of 53000. Electrophoresis of sodium-dodecyl-sulfate-solubilized yeast homogenate followed by immune replica analysis showed that the value of 53000 is the Mr of a non-degraded form. The purified enzyme had an isoelectric point of 5.3 and a pH optimum of 7.4. Pyridoxal 5'-phosphate has been shown to be an essential cofactor. The enzyme activity was sensitive to thiol blocking reagents. Hemin, but not heme, inhibited the activity of the purified enzyme.

Relation of the extra-sequence of the precursor form of chicken liver delta-aminolevulinate synthase to its quaternary structure and catalytic properties.

Precursor and mature forms of delta-aminolevulinate (ALA) synthase were purified to near homogeneity from chicken liver mitochondria and cytosol, respectively, and their properties were compared. The enzyme purified from mitochondria had apparently the same subunit molecular weight (65,000) as that of the native mitochondrial enzyme. The enzyme purified from the cytosol fraction, however, showed a subunit molecular weight of about 71,000 which was somewhat smaller than that estimated for the native cytosolic enzyme (73,000). The enzyme purified from liver cytosol seems to have been partially degraded by some endogenous protease during the purification, but may have the major part of the signal sequence. On sucrose density gradient centrifugation, the purified mitochondrial and cytosolic ALA synthases showed an apparent molecular weight of about 140,000, indicating that both enzymes exist in a dimeric form. The ALA synthase synthesized in vitro was also shown to exist as a dimer. Apparently the extra-sequence does not interfere with the formation of dimeric form of the enzyme. The purified cytosolic ALA synthase had a specific activity comparable to that of the purified mitochondrial enzyme. Kinetic properties of the two enzymes, such as the pH optimum and the apparent Km values for glycine and succinyl-CoA, were quite similar. The extra-sequence does not appear to affect the catalytic properties of ALA synthase. The isoelectric point of the cytosolic ALA synthase was 7.5, whereas that of the mitochondrial enzyme was 7.1. This suggests that the extra-sequence in the cytosolic enzyme may be relatively rich in basic amino acids.

delta-Aminolevulinate synthase isozymes in the liver and erythroid cells of chicken.

Antibodies raised against the purified chicken liver delta-aminolevulinate synthase showed a partial cross-reactivity with the chicken erythroid delta-aminolevulinate synthase. delta-Aminolevulinate synthase synthesized in vitro using polysomes from erythroid cells showed a subunit molecular weight of 55,000, whereas the enzyme synthesized in vitro using liver polysomes had a subunit molecular weight of 73,000. delta-Aminolevulinate synthase isolated from mitochondria of erythroid cells showed a molecular weight of 53,000, while the enzyme in liver mitochondria had a value of 65,000. These observations imply that the erythroid delta-aminolevulinate synthase differs from the hepatic enzyme.

Aging-related decreases in hepatic mitochondrial and cytosolic delta-aminolevulinic acid synthase during experimental porphyria.

The basal- and allylisopropylacetamide-induced activities of the first enzyme of heme biosynthesis, delta-aminolevulinic acid synthase (ALAS) were measured in hepatic mitochondria and cytosol of young, adult, and aged Fisher 344 rats. The total cellular ALAS activity induced by allylisopropylacetamide decreased 67% with age. The specific activity of mitochondrial ALAS in normal and induced animals decreased with aging when assayed in whole or broken mitochondria. The levels of ALAS which accumulated in the cytosol after allylisopropylacetamide administration were proportionally greater in both the young and senescent than in the mature animals. During aging, no evidence for a fragile population of mitochondria in either normal or induced animals was observed suggesting that mitochondrial matrix proteins are not released during homogenization. The hepatic mitochondrial content decreased during aging when calculated using both a membrane-bound marker enzyme cytochrome oxidase and a matrix marker enzyme citrate synthase and was unaffected by allylisopropylacetamide treatment. This reduced mitochondrial content further diminishes the level of functional ALAS available in the liver during senescence. This study confirms the age-dependent decrease in mitochondria ALAS in normal and induced animals and also suggests an age-related change in the process by which cytosolic ALAS is translocated into the mitochondria.

[Various properties of delta-aminolevulinate synthetase from Rhodopseudomonas palustris]. / Algunas propiedades de la delta-aminolevulinato sintetasa de Rhodopseudomonas palustris.

The enzymatic formation of delta-aminolevulinic acid (ALA) in extracts of Rhodopseudomonas palustris has been studied. Some properties of delta-aminolevulinate synthetase (ALA-S), including molecular weight km for glycine and the inhibition of enzyme activity by ALA are reported.

Purification of 5-aminolaevulinate synthase from liver mitochondria of chick embryo.

5-Aminolaevulinate synthase from chick-embryo liver mitochondria has, for the first time, been purified to homogeneity in its native non-degraded form by molecular sieve chromatography, chromatofocusing and affinity chromatography. The enzyme has a minimum molecular weight of 68000 as determined by sodium dodecylsulphate/polyacrylamide gel electrophoresis and a specific activity of 35000 units/mg of protein. This result conflicts with the previous report of Whiting, M.J. and Granick, G. [(1976) J. Biol. Chem. 251, 1340-1346] that the chick embryo enzyme has a molecular weight of 49000. We show here that the purified form can be degraded proteolytically to a smaller form of molecular weight around 50000 while retaining full enzymatic activity. It seem evident, therefore, that the enzyme isolated by Whiting & Granick (1976) was degraded. We have further established by pulse-labelling studies and immunoprecipitation that the enzyme isolated by our new and rapid procedure has the same minimum molecular weight as that which exists in vivo.