Knockout of secondary alcohol dehydrogenase in Nocardia cholesterolicum NRRL 5767 by CRISPR/Cas9 genome editing technology.

Nocardia cholesterolicum NRRL 5767 is well-known for its ability to convert oleic acid to 10-hydroxystearic acid (~88%, w/w) and 10-ketostearic acid (~11%, w/w). Conversion of oleic acid to 10-hydroxystearic acid and then to 10-ketostearic acid has been proposed to be catalyzed by oleate hydratase and secondary alcohol dehydrogenase, respectively. Hydroxy fatty acids are value-added with many industrial applications. The objective of this study was to improve the Nocardia cholesterolicum NRRL5767 strain by CRISPR/Cas9 genome editing technology to knockout the secondary alcohol dehydrogenase gene, thus blocking the conversion of 10-hydroxystearic acid to 10-ketostearic acid. The improved strain would produce 10-hydroxystearic acid solely from oleic acid. Such improvement would enhance the production of 10-hydroxystearic acid by eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. Here, we report: (1) Molecular cloning and characterization of two functional recombinant oleate hydratase isozymes and a functional recombinant secondary alcohol dehydrogenase from Nocardia cholesterolicum NRRL5767. Existence of two oleate hydratase isozymes may explain the high conversion yield of 10-hydroxystearic acid from oleic acid. (2) Construction of a CRISPR/Cas9/sgRNA chimeric plasmid that specifically targeted the secondary alcohol dehydrogenase gene by Golden Gate Assembly. (3) Transformation of the chimeric plasmid into Nocardia cholesterolicum NRRL 5767 by electroporation and screening of secondary alcohol dehydrogenase knockout mutants. Two mutants were validated by their lack of secondary alcohol dehydrogenase activity at the protein level and mutation at the targeted 5' coding region and the 5' upstream at the DNA level. The knockout mutants offer improvements by converting added oleic acid to solely 10-hydroxystearic acid, thus eliminating downstream separation of 10-hydroxystearic acid from 10-ketostearic acid. To the best of our knowledge, we report the first successful knockout of a target gene in the Nocardia species using CRISPR/Cas9/sgRNA-mediated genome editing technology.

Bayes-Like Integration of a New Sensory Skill with Vision.

Humans are effective at dealing with noisy, probabilistic information in familiar settings. One hallmark of this is Bayesian Cue Combination: combining multiple noisy estimates to increase precision beyond the best single estimate, taking into account their reliabilities. Here we show that adults also combine a novel audio cue to distance, akin to human echolocation, with a visual cue. Following two hours of training, subjects were more precise given both cues together versus the best single cue. This persisted when we changed the novel cue's auditory frequency. Reliability changes also led to a re-weighting of cues without feedback, showing that they learned something more flexible than a rote decision rule for specific stimuli. The main findings replicated with a vibrotactile cue. These results show that the mature sensory apparatus can learn to flexibly integrate new sensory skills. The findings are unexpected considering previous empirical results and current models of multisensory learning.

Modulation of nitrosative stress via glutathione-dependent formaldehyde dehydrogenase and S-nitrosoglutathione reductase.

Glutathione-dependent formaldehyde dehydrogenase (GFD) from Taiwanofungus camphorata plays important roles in formaldehyde detoxification and antioxidation. The enzyme is bifunctional. In addition to the GFD activity, it also functions as an effective S-nitrosoglutathione reductase (GSNOR) against nitrosative stress. We investigated the modulation of HEK (human embryonic kidney) 293T cells under nitrosative stress by transfecting a codon optimized GFD cDNA from Taiwanofungus camphorata (Tc-GFD-O) to these cells. The parental and transfected HEK 293T cells were then subjected to S-nitrosoglutathione treatment to induce nitrosative stress. The results showed that in Tc-GFD-O-transfected 293T cells, the expression and activity of GFD increased. Additionally, these cells under the nitrosative stress induced by S-nitrosoglutathione showed both higher viability and less apoptosis than the parental 293T cells. This finding suggests that the Tc-GFD-O in HEK 293T cells may provide a protective function under nitrosative stress.

A peroxiredoxin cDNA from Taiwanofungus camphorata: role of Cys31 in dimerization.

Peroxiredoxins (Prxs) play important roles in antioxidant defense and redox signaling pathways. A Prx isozyme cDNA (TcPrx2, 745 bp, EF552425) was cloned from Taiwanofungus camphorata and its recombinant protein was overexpressed. The purified protein was shown to exist predominantly as a dimer by sodium dodecyl sulfate-polyacrylamide gel electrolysis in the absence of a reducing agent. The protein in its dimeric form showed no detectable Prx activity. However, the protein showed increased Prx activity with increasing dithiothreitol concentration which correlates with dissociation of the dimer into monomer. The TcPrx2 contains two Cys residues. The Cys(60) located in the conserved active site is the putative active peroxidatic Cys. The role of Cys(31) was investigated by site-directed mutagenesis. The C31S mutant (C(31) → S(31)) exists predominantly as a monomer with noticeable Prx activity. The Prx activity of the mutant was higher than that of the corresponding wild-type protein by nearly twofold at 12 µg/mL. The substrate preference of the mutant was H2O2 > cumene peroxide > t-butyl peroxide. The Michaelis constant (K M) value for H2O2 of the mutant was 0.11 mM. The mutant enzyme was active under a broad pH range from 6 to 10. The results suggest a role of Cys(31) in dimerization of the TcPrx2, a role which, at least in part, may be involved in determining the activity of Prx. The C(31) residue does not function as a resolving Cys and therefore the TcPrx2 must follow the reaction mechanism of 1-Cys Prx. This TcPrx2 represents a new isoform of Prx family.

Biochemical characterization of a functional recombinant aryl-alcohol dehydrogenase from Taiwanofungus camphorata.

BACKGROUND: Aryl-alcohol dehydrogenases (AADs) have been known to involve in the metabolism of aromatic compounds. RESULTS: One TcAAD cDNA (GenBank HQ453361) encoding a putative aryl-alcohol dehydrogenase (AAD) was cloned from Taiwanofungus camphorata. The deduced amino acid sequence is conserved among the reported AADs. A 3-D structural model of the TcAAD has been created based on the known structure of voltage-dependent potassium channels subunit beta-2 (PDB code: 3EAU). To characterize the TcAAD, the coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His6-tagged TcAAD was overexpressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 39 kDa on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 40.58 kDa which suggests that the purified enzyme is a monomeric enzyme. Using veratraldehyde as a substrate, the KM, Vmax of TcADD was determined at pH 6.0. Using benzyl alcohol derivatives as substrates, the oxidizing power of TcADD via NAD+ at pH 9.6 was studied. CONCLUSIONS: The coding sequence of the TcAAD cDNA was introduced into an S. cerevisiae expression system and the active enzyme purified and characterized. Understanding the properties of this TcAAD will be beneficial for its potential in xenobiotic detoxification or production of natural flavors.

Molecular cloning and characterization of a thioredoxin from Taiwanofungus camphorata.

BACKGROUND: Thioredoxin (Trx) is reduced by thioredoxin reductase. Trx is used in ribonucleoide reduction, assimilatory sulfate reduction, in modulation of protein sulfhydryl groups, and refolding proteins. RESULTS: A TcTrx (Tc: Taiwanofungus camphorata) cDNA (640 bp, GenBank AY838902.1) encoding a putative thioredoxin (Trx) of 135 amino acid residues with calculated molecular mass of 16.17 kDa was cloned from Taiwanofungus c amphorata. The deduced amino acid sequence containing a motif (Cys-Gly-Pro-Cys) that is highly conserved among the reported Trxs. A three dimensional structural model of the TcTrx has been created based on the known structure of Malassezia sympodialis Trx (MsTrx, PDB ID: 2j23). To characterize the TcTrx, the codon optimized coding region was subcloned into an expression vector and transformed into Saccharomyces cerevisiae. The recombinant His8-tagged TcTrx was expressed and purified by Ni affinity chromatography. The purified enzyme showed a band of approximately 32 kDa (expected dimeric form) on a 12% SDS-PAGE. The molecular mass determined by MALDI-TOF is 33.16 kDa which suggests that the purified enzyme is a dimeric enzyme. Furthermore, the enzyme exhibited TcTrx activity via insulin assay. The Michaelis constant (K M ) value for insulin was 3.78 × 10-2 mM. The enzyme's half-life of deactivation was 13 min at 45°C. The enzyme was most active at pH 7. CONCLUSIONS: A three dimensional structural model of T. camphorata Trx based on its TcTrx cDNA sequence. The active form of the TcTrx has been successfully expressed in yeast. The enzyme possesses Trx activity and is capable of reduction of disulfide bonds during the formation of newly synthesized proteins.

Lemon protein disulfide isomerase: cDNA cloning and biochemical characterization.

BACKGROUND: Protein disulfide isomerases (PDIs), a family of structurally related enzymes, aid in protein folding by catalyzing disulfide bonds formation, breakage, or isomerization in newly synthesized proteins and thus. RESULTS: A ClPDI cDNA (1828 bp, GenBank accession HM641784) encoding a putative PDI from Citrus limonum was cloned by polymerase chain reaction (PCR). The DNA sequence encodes a protein of 500 amino acids with a calculated molecular mass of 60.5 kDa. The deduced amino acid sequence is conserved among the reported PDIs. A 3-D structural model of the ClPDI has been created based on the known crystal structure of Homo sapiens (PDB ID: 3F8U_A). The enzyme has two putative active sites comprising the redox-active disulfides between residues 60-63 and 405-408 (motif CGHC). To further characterize the ClPDI, the coding region was subcloned into an expression vector pET-20b (+), transformed into E. coli Rosetta (DE3)pLysS, and recombinant protein expressed. The recombinant ClPDI was purified by a nickel Sepharose column. PDI's activity was assayed based on the ability of the enzyme to isomerize scrambled RNase A (sRNase A) to active enzyme. The KM, kcat and kcat/KM values were 8.3 × 10-3 µM, 3.0 × 10-5 min-1, and 3.6 × 10-1 min-1 mM-1. The enzyme was most active at pH 8. CONCLUSIONS: The advantage of this enzyme over the PDI from all other sources is its low KM. The potential applications of this PDI in health and beauty may worth pursuing.

Taiwanofungus camphorata nitroreductase: cDNA cloning and biochemical characterisation.

Nitroreductases (Nrs) play important roles in redox system via NADPH or NADH as a reductant. A TcNr cDNA encoding a putative Nr was cloned from Taiwanofungus camphorata. A 3-D structural model of the TcNr has been created based on the known structure of BcNr (Bacillus cereus). To characterise the TcNr, the coding region was subcloned into an expression vector and transformed into Escherichia coli. The recombinant His(6)-tagged TcNr was purified by Ni affinity chromatography. The purified enzyme showed a single band at molecular mass of approximately 25 kDa on 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The enzyme exhibited Nr activity via ferricyanide assay. The Michaelis constant (K(M)) value for ferricyanide was 0.86 mM. The enzyme(')s half-life of deactivation at 45°C was 12.3 min. The enzyme was most active at pH 6. The enzyme's preferred substrate is 1-chloro-2, 4-dinitrobenzene.

Monothiol glutaredoxin cDNA from Taiwanofungus camphorata: a novel CGFS-type glutaredoxin possessing glutathione reductase activity.

Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzyme's half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.

Cloning, expression, and characterization of a thioredoxin reductase cDNA from Taiwanofungus camphorata.

A cDNA encoding putative thioredoxin reductase (TR) was identified from a medicinal mushroom, Taiwanofungus camphorata (T. camphorata). Alignment of the deduced amino acid sequence with TRs from other organisms showed high levels of identity (59-74%). A three-dimensional (3-D) homology structure was created for this TR. Functional T. camphorata TR (TcTR) was overexpressed in yeast and purified. The purified enzyme showed a monomic form on a 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme's half-life of deactivation at 60 degrees C was 12.9 min, and its thermal inactivation rate constant K(d) was 5.37 x 10(-2) min(-1). The optimal pH for the enzyme was pH 8 and retained about 76% activity in the presence of 0.1 M imidazole. The enzyme showed 50% activity after 10 min of incubation at 37 degrees C with chymotrypsin. The Michaelis constant (K(m)) value for dithionitrobenzoate (DTNB) was 1.59 mM.

Mutagenicity evaluation of metal oxide nanoparticles by the bacterial reverse mutation assay.

Nanomaterials have been emerging as a new group of contaminants in the environment. We reported the use of a bacterial reverse mutation assay (Ames assay) to evaluate the mutagenicity of five metal oxide nanoparticles Al(2)O(3), Co(3)O(4), CuO, TiO(2), and ZnO in this study. Results showed the mutagenicity was negative for four nanoparticles (Al(2)O(3), Co(3)O(4), TiO(2), and ZnO) up to 1000mug/plate to all three tested strains without S9 metabolic activation. Using a preincubation procedure and high S9 (9%) activation, TiO(2) and ZnO induced marginal mutagenesis to strain Escherichia coli WP2 trp uvrA. CuO displayed low mutagenic potential to Salmonella typhimurium TA97a and TA100 at specific concentrations. However, the colony inhibition effect of CuO was predominant to the strain E. coli WP2 trp uvrA. A dose-dependent inhibition of Escherichia coli WP2 colony was found under CuO exposure at concentration range of 100-1600mug/plate. No growth inhibition of tested bacterial strains by Al(2)O(3), Co(3)O(4), and ZnO was observed at the concentrations used.

Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from Taiwanofungus camphorata.

Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli . Functional TcGrx was expressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of approximately 15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (K(m)) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K(d) was 6.52 x 10(-2) min(-1). The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.

Functional expression of Francisella tularensis FabH and FabI, potential antibacterial targets.

Francisella tularensis is an extremely infectious airborne pathogen that has long been considered as a potential biological weapon. Enzymes of fatty acid synthesis (FAS) pathway are attractive targets for the development of new antibacterial agents because of differences between the biosynthesis pathways of bacteria and mammals. We report here the first expression of three functional enzymes in F. tularensis FAS-II pathway: FabH (3-oxoacyl-acyl carrier protein synthase III) which initiates elongation in FAS-II; FabD (Malonyl-CoA-acyl carrier protein transacylase) which catalyzes the transfer of a malonyl moiety from malonyl-CoA to ACP generating malonyl-ACP, and FabI (enoyl-ACP reductase) which catalyzes the reduction of enoyl-acyl-ACP derivatives. The genes encoding the FabD, FabH, and FabI were custom synthesized and cloned in pET15b expression vector. Each recombinant His-tagged fusion protein was overexpressed by IPTG induction, and then purified by affinity chromatography on a Ni-NTA column. The purified FabH and FabI have been used as targets for new drug development. Screening of a class of indole-2-carboxylic acid compounds has led to the discovery of several new compounds with promising activity against F. tularensis FabH or FabI enzymes. For example, indole derivative WIUAKP-001 inhibited 80% the FabH enzyme at 40 microM with IC(50) value of 2 microM whereas WIUAKP-031 inhibited 98% the FabI enzyme at 37.5 microM with IC(50) value of 6 microM. These compounds hold great promise for future development of new indole derivatives as inhibitors of type II FAS enzymes, and as potential new treatment for tularemia.

Production of 10-hydroxy-8(E)-octadecenoic acid from oleic acid conversion by strains of Pseudomonas aeruginosa.

Eighteen Pseudomonas aeruginosa strains were examined for their ability to convert oleic acid to produce 10-hydroxy-8(E)-octadecenoic acid (HOD), which was structurally confirmed by GC-MS, NMR, and FTIR. There were no substantial amounts of other new compounds found in the fermentation broths in addition to HOD and 7,10-dihydroxy-8(E)-octadecenoic acid (DOD). The results demonstrated that P. aeruginosa strains possessed varying levels of activity for producing HOD. Under the experimental conditions, strain NRRL B-14938 isolated from sheep manure was the best HOD producer exhibiting the highest HOD to DOD product ratio in the medium most suitable for purifying HOD. Using strain B-14938 as a model system for further characterization, optimum conditions for producing HOD were found to be at 26 degrees C and pH 7.0 after 60 h of reaction time using a medium containing EDTA as a chelating agent. This study has identified a high-yielding P. aeruginosa strain and provided the reaction characteristics needed to develop a scale-up production process of HOD for testing its properties and potential new uses.

Dehydroascorbate reductase cDNA from sweet potato (Ipomoea batatas [L.] Lam): expression, enzyme properties, and kinetic studies.

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The protein's half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.

A highly stable cambialistic-superoxide dismutase from Antrodia camphorata: expression in yeast and enzyme properties.

A cDNA encoding a putative superoxide dismutase (SOD) was identified in expressed sequence tags of Antrodia camphorata, a medicinal mushroom found only in Taiwan. The deduced protein was aligned with Mn-SODs and Fe-SODs from other organisms, this SOD showed greater homology to Mn-SOD. Functional A. camphorata SOD protein was overexpressed in yeast and purified. The purified enzyme showed two active forms on a 12.5% native PAGE, a dimer and a monomer. The dimeric protein's half-life of deactivation at 80 degrees C was 7 min, and its thermal inactivation rate constant K(d) was 9.87 x 10(-2)min(-1). The enzyme was stable in a broad pH range from 5-11; in the presence of 0.4M imidazole and 2% SDS. The atomic absorption spectrometric assay showed that 1.0 atom of manganese/iron (9:1) was present in each SOD subunit. The high stability of the enzyme make it better suited than other cambialistic-SODs for use in cosmetics. The SOD also documents its future utility in developing anti-inflammatory agent and in the treatment of chronic diseases.

Molecular cloning and functional expression of bovine deoxyhypusine hydroxylase cDNA and homologs.

Deoxyhypusine hydroxylase is the second of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here, we describe the cloning and characterization of bovine deoxyhypusine hydroxylase cDNA and its homologs. The deduced bovine deoxyhypusine hydroxylase protein is 87% identical to human enzyme and 45% identical to yeast enzyme. The overexpressed enzyme showed activity in catalyzing the hydroxylation of the deoxyhypusine residue in the eIF5A intermediate. An amino acid substitution from Glu 57 to Gly located at one of the four conserved His-Glu (HE) pairs, the potential metal coordination sites, resulted in severe reduction of deoxyhypusine hydroxylase activity. A deletion at the HEAT-repeats 1-3 resulted in complete losses of deoxyhypusine hydroxylase activity.

Biochemical characterization of 1-Cys peroxiredoxin from Antrodia camphorata.

Antrodia camphorata is a unique medicinal mushroom found only in Taiwan. It has been used as a remedy for various diseases in folk medicine. Antrodia camphorata has been shown to exhibit antioxidative effects. Peroxiredoxins play important roles in antioxidation and cell signaling. A gene encoding an antioxidant enzyme, 1-cysteine peroxiredoxin (1-Cys Prx), was identified in an expressed sequence tag database of the A. camphorata and cloned by polymerase chain reaction. The 1-Cys Prx cDNA (837 bp, accession no. AY870325) contains an open reading frame encoding a protein of 223 amino acid residues with calculated molecular mass of 25,081 Da. The deduced protein shared 44-58% identity with 1-Cys Prx from Homo sapiens, Bos taurus, and Saccharomyces cerevisia. The sequence surrounding the conserved cysteine DFTPVCTTE is conserved. The coding sequence was subcloned into a vector, pET-20b (+), and transformed into Escherichia coli. The recombinant 1-Cys Prx was purified by Ni(2+)-nitrilotriacetic acid (Sepharose). The purified enzyme was characterized under various conditions. The enzyme is thermostable because its half-life of inactivation was 15.5 min at 60 degrees C. It was stable under alkaline pH range from 7.8 to 10.2. The enzyme showed decreased activity with increasing concentration of imidazole. The enzyme is sensitive to trypsin and chymotrypsin treatment.

Biochemical characterization of a cambialistic superoxide dismutase isozyme from diatom Thallassiosira weissflogii: cloning, expression, and enzyme stability.

A cDNA clone of 1081 bp encoding a second putative superoxide dismutase (SOD) from diatom Thallassiosira weissflogii was cloned by the polymerase chain reaction technique. The cDNA encodes a protein of 286 amino acid residues. Alignment of the truncated SOD sequence containing 217 amino acid residues with Mn-SODs from Vibrio mimicus and Escherichia coli, as well as two Fe-SODs from E. coli and Photobacterium leiognathi, this SOD showed greater homology to Mn-SOD. The residues required to coordinate the manganese ion were conserved in all reported Mn-SOD. The recombinant SOD has a half life of deactivation of 14.7 min at 65 degrees C. Its thermal inactivation rate constant Kd was 3.21 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 4 to 12. The presence of imidazole (up to 0.8 M) and sodium dodecylsulfate (up to 4%) had little effect on the enzyme's activity. The atomic absorption spectrometric assay showed the presence of 0.3 atom of iron/manganese (2:1) in each SOD subunit. Reconstituted activity suggested that diatom SOD was cambialistic Fe/Mn-SOD.

Molecular cloning of bovine eIF5A and deoxyhypusine synthase cDNA.

Deoxyhypusine synthase is the first of the two enzymes that catalyzes the maturation of eukaryotic initiation factor 5A (eIF5A). The mature eIF5A is the only known protein in eukaryotic cells that contains the unusual amino acid hypusine (N(epsilon)-(4-amino-2(R)-hydroxybutyl)-lysine). Synthesis of hypusine is essential for the function of eIF5A in eukaryotic cell proliferation and survival. Here we describe the cloning and characterization of bovine eIF5A and bovine deoxyhypusine synthase. The deduced bovine eIF5A protein is 100% identical to human eIF5A-1, and the deduced bovine deoxyhypusine synthase protein showed a 93% identity to the human protein.