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S Swaminathan

Publications and source records attributed to S Swaminathan.

At least 19 recordsLinked to original sources
PubMed2001

Crystallographic evidence for doxorubicin binding to the receptor-binding site in Clostridium botulinum neurotoxin B.

The neurotoxins of Clostridium botulinum and tetanus bind to gangliosides as a first step of their toxin activity. Identifying suitable receptors that compete with gangliosides could prevent toxin binding to the neuronal cells. A possible ganglioside-binding site of the botulinum neurotoxin B (BoNT/B) has already been proposed and evidence is now presented for a drug binding to botulinum neurotoxin B from structural studies. Doxorubicin, a well known DNA intercalator, binds to the neurotoxin at the receptor-binding site proposed earlier. The structure of the BoNT/B-doxorubicin complex reveals that doxorubicin has interactions with the neurotoxin similar to those of sialyllactose. The aglycone moiety of the doxorubicin stacks with tryptophan 1261 and interacts with histidine 1240 of the binding domain. Here, the possibility is presented of designing a potential antagonist for these neurotoxins from crystallographic analysis of the neurotoxin-doxorubicin complex, which will be an excellent lead compound.

Bacterial Proteins↗
PubMed2001

Structure of staphylococcal enterotoxin C2 at various pH levels.

The three-dimensional structure of staphylococcal enterotoxin C2 (SEC2), a toxin as well as a superantigen, has been determined at various pH levels from two different crystal forms, tetragonal (pH 5.0, 5.5, 6.0 and 6.5) and monoclinic (pH 8.0) at 100 and 293 K, respectively, by the molecular-replacement method. Tetragonal crystals belong to space group P4(3)2(1)2, with unit-cell parameters a = b = 42.68, c = 289.15 A (at pH 5.0), and monoclinic crystals to space group P2(1), with unit-cell parameters a = 43.3, b = 70.6, c = 42.2 A, beta = 90.3 degrees. SEC2 contains a zinc-binding motif, D+HExxH, and accordingly a Zn atom has been identified. The coordination of the zinc ion suggests that it may be catalytic zinc rather than structural, but there is so far no biological evidence that it possesses catalytic activity. However, superantigen staphylococcal exfoliative toxins A and B have been shown to have enzymatic activity after their fold was identified to be similar to that of serine protease. The structure and its conformation are similar to the previously reported structures of SEC2. Though it was expected that the zinc ion may be leached out, as the histidines coordinating the zinc ion are expected to be protonated below pH 6.0, zinc is present at all pH values. The coordination distances to zinc increase with decreasing pH, with the distances being the least at pH 8.0. The results of automated model building using the ARP/wARP program for different data sets collected at various pH values are discussed.

Bacterial Proteins↗
PubMed2001

Expression of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris using the GAP promoter.

High-level expression and efficient assembly of Hepatitis B surface Antigen (HBsAg) particles have been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under the control of the alcohol oxidase (AOX1) promoter. However, the time taken to reach peak product concentration is usually very long ( approximately 240 h). In this paper, we describe the expression of HBsAg in P. pastoris using the recently described glyceraldehyde-3-phosphate dehydrogenase (GAP) promoter. Unlike the previously described AOX1 promoter based system (in which biomass is generated first followed by methanol-induced antigen production), biomass generation and antigen production occur simultaneously in medium containing glycerol or glucose. Maximal levels of HBsAg expression in case of the single copy AOX1 integrant (attained after 6 days of induction) exceeded the levels of antigen produced by the single copy GAP integrant. However, this was offset by continuous antigen production by the GAP clone. In an attempt to further enhance antigen production levels of the GAP clones, we isolated multicopy Pichia integrants containing up to four copies of the GAP promoter-driven constitutive expression cassette using the Zeocin screening procedure. The data demonstrated a direct correlation between the gene dosage and the levels of HBsAg expressed by the GAP clones. The effect of copy number was additive and the four copy clone resulted in about four-fold higher yield of HBsAg. The majority of HBsAg produced in the constitutive expression system was found to be of particulate form, based on sedimentation behaviour and particle-specific ELISA, suggesting that it has the potential to serve as an effective immunogen. These particles were sensitive to thiol reagents. We also explored the possibility of secreting the GAP expressed HBsAg in P. pastoris. In-frame fusion of the Saccharomyces cerevisiae alpha-factor secretion signal under the constitutive GAP promoter resulted in secretion of approximately 20 nm HBsAg particles as evidenced by electron microscopy. However, the levels of secreted HBsAg particles were very low, presumably due to the inherent hydrophobicity of the HBsAg molecule and the consequent propensity for membrane association. Our studies show that secretion is not a good strategy for expression of HBsAg in P. pastoris. The data also suggests that intracellular production of HBsAg under the GAP promoter using multicopy expression cassettes can indeed serve as an effective alternative to the AOX1 promoter. Further, the GAP promoter based system obviates the need to use and extensively monitor methanol during recombinant antigen production. Finally, this constitutive system has the potential for continuous culture wherein several batches of recombinant protein-containing biomass can be harvested from a single initial fermentation.

Alcohol Oxidoreductases↗
PubMed2001

Crystallization and preliminary X-ray studies of meso-2,3-butanediol dehydrogenase from Klebsiella pneumoniae IAM1063.

Meso-2,3-butanediol dehydrogenase (meso-BDH) has been crystallized and preliminary X-ray crystallographic characterization of meso-BDH crystals has been performed. Single crystals of meso-BDH were prepared in two forms by the hanging-drop vapour-diffusion method using polyethylene glycol as a precipitant. Form I crystals belong to space group C2, with unit-cell parameters a = 215.5, b = 79.4, c = 134.8 A, beta = 98.22 degrees, and form II crystals belong to space group P2(1), with unit-cell parameters a = 69.16, b = 109.78, c = 127.28 A, beta = 102.29 degrees. The crystals diffracted to 2.0 and 1.7 A resolutions, respectively, using synchrotron radiation.

Alcohol Oxidoreductases↗
PubMed2001

Interaction of human pancreatic ribonuclease with human ribonuclease inhibitor. Generation of inhibitor-resistant cytotoxic variants.

Mammalian ribonucleases interact very strongly with the intracellular ribonuclease inhibitor (RI). Eukaryotic cells exposed to mammalian ribonucleases are protected from their cytotoxic action by the intracellular inhibition of ribonucleases by RI. Human pancreatic ribonuclease (HPR) is structurally and functionally very similar to bovine RNase A and interacts with human RI with a high affinity. In the current study, we have investigated the involvement of Lys-7, Gln-11, Asn-71, Asn-88, Gly-89, Ser-90, and Glu-111 in HPR in its interaction with human ribonuclease inhibitor. These contact residues were mutated either individually or in combination to generate mutants K7A, Q11A, N71A, E111A, N88R, G89R, S90R, K7A/E111A, Q11A/E111A, N71A/E111A, K7A/N71A/E111A, Q11A/N71A/E111A, and K7A/Q11A/N71A/E111A. Out of these, eight mutants, K7A, Q11A, N71A, S90R, E111A, Q11A/E111A, N71A/E111A, and K7A/N71A/E111A, showed an ability to evade RI more than the wild type HPR, with the triple mutant K7A/N71A/E111A having the maximum RI resistance. As a result, these variants exhibited higher cytotoxic activity than wild type HPR. The mutation of Gly-89 in HPR produced no change in the sensitivity of HPR for RI, whereas it has been reported that mutating the equivalent residue Gly-88 in RNase A yielded a variant with increased RI resistance and cytotoxicity. Hence, despite its considerable homology with RNase A, HPR shows differences in its interaction with RI. We demonstrate that interaction between human pancreatic ribonuclease and RI can be disrupted by mutating residues that are involved in HPR-RI binding. The inhibitor-resistant cytotoxic HPR mutants should be useful in developing therapeutic molecules.

Amino Acid Substitution↗
PubMed2001

Crystal structure of outer surface protein C (OspC) from the Lyme disease spirochete, Borrelia burgdorferi.

Outer surface protein C (OspC) is a major antigen on the surface of the Lyme disease spirochete, Borrelia burgdorferi, when it is being transmitted to humans. Crystal structures of OspC have been determined for strains HB19 and B31 to 1.8 and 2.5 A resolution, respectively. The three-dimensional structure is predominantly helical. This is in contrast to the structure of OspA, a major surface protein mainly present when spirochetes are residing in the midgut of unfed ticks, which is mostly beta-sheet. The surface of OspC that would project away from the spirochete's membrane has a region of strong negative electrostatic potential which may be involved in binding to positively charged host ligands. This feature is present only on OspCs from strains known to cause invasive human disease.

Amino Acid Sequence↗
PubMed2001

Rapid engineering of bacterial artificial chromosomes using oligonucleotides.

A rapid method obviating the use of selectable markers to genetically manipulate large DNA inserts cloned into bacterial artificial chromosomes is described. Mutations such as single-base changes, deletions, and insertions can be recombined into a BAC by using synthetic single-stranded oligonucleotides as targeting vectors. The oligonucleotides include the mutated sequence flanked by short homology arms of 35-70 bases on either side that recombine with the BAC. In the absence of any selectable marker, modified BACs are identified by specific PCR amplification of the mutated BAC from cultures of pooled bacterial cells. Each pool represents about 10 electroporated cells from the original recombination mixture. Subsequently, individual clones containing the desired alteration are identified from the positive pools. Using this BAC modification method, we have observed a frequency of one recombinant clone per 90-260 electroporated cells. The combination of high targeting frequency and the sensitive yet selective PCR-based screening method makes BAC manipulation using oligonucleotides both rapid and simple.

Bacterial Proteins↗
PubMed2001

DNA damage in human transitional cell carcinoma cells after exposure to the proximate metabolite of the bladder carcinogen 4-aminobiphenyl.

The DNA damage induced by N-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP), was examined in human transitional cell carcinoma (TCC) cells after exposure to the chemical in vitro. 32P-postlabeling analysis of TCC cultures exposed to N-OH-AABP revealed a minor adduct identified as 3-(deoxyguanosin-N2-yl)-4-acetylaminobiphenyl (dG-N2-AABP) based on comparison of the HPLC and TLC mobility of the product with the synthetic standard. An adduct with the same chromatographic properties was also detected on postlabeling analyses of calf thymus DNA bound to N-OH-AABP by incubation with horseradish peroxidase and hydrogen peroxide. Detection of dG-N2-AABP, which contains the acetyl moiety, suggests that N-acetoxy-4-acetylamino-biphenyl might be formed as a reactive intermediate and could conceivably arise by a free-radical-mediated reaction of N-OH-AABP with endogenous peroxidases. The radical intermediates could also form reactive oxygen species (ROS). To test this possibility, TCC cultures were exposed to N-OH-AABP and the formation of ROS was measured using 2,7-dichlorofluorescein (DCF) fluorescence assay. TCC cultures exposed to N-OH-AABP showed a dose-dependent increase in the ratio of DCF/DNA fluorescence compared to the untreated controls. Formation of ROS was inhibited by butylated hydroxyanisole (BHA). Furthermore, oxidative DNA damage resulting from ROS was monitored by measurement of 8-oxoguanine products by immunochemical staining and the TCC cells treated with N-OH-AABP revealed a characteristic staining. These results suggest that N-OH-AABP caused oxidative DNA damage as well as bulky covalent adducts in urothelial DNA, possibly involving endogenous peroxidases. These findings show that human uroepithelial cells, which are the target cell types in vivo for arylamine-induced cancers, are metabolically capable of activating these proximate carcinogenic metabolites of arylamines, and these reactions might play a determinate role in the genotoxicity of these environmental carcinogens.

Aminobiphenyl Compounds↗
PubMed2001

Effect of copy number on the expression levels of hepatitis B surface antigen in the methylotrophic yeast Pichia pastoris.

High-level expression and efficient assembly of hepatitis B surface Antigen (HBsAg) has been reported in Pichia pastoris by integrating a single copy of the HBsAg gene under control of the AOX1 promoter. To fully utilize the expression potential of the P. pastoris expression system, we investigated the influence of gene copy number on the expression of HBsAg in this yeast. A panel of Pichia clones carrying progressively increasing copies of the heterologous gene expression cassette was created using an in vitro multimerization approach. Using this strategy, constructs containing up to a maximum of eight direct repeats of the HBsAg-expressing cassettes could be created. These expression cassettes were targeted for integration into the genome of the host strain GS115 with simultaneous elimination of the resident AOX1 gene. Deletion of the AOX1 gene was intended to create Mut(s) (methanol utilization slow) transformants that are known to have an increased ability to generate HBsAg in particulate form. A systematic investigation of the resultant clones demonstrated that the increase in copy number results in a proportional elevation in the steady-state levels of the HBsAg-specific mRNA, which in turn is closely paralleled by a corresponding increase in the total levels of the HBsAg protein. Virtually all the recombinant protein in the soluble fraction was present in the particulate form based on particle-specific ELISA and sedimentation behavior. Further, our studies also revealed the continued physical and functional integrity of the HBsAg-expressing cassettes during the course of an extended induction phase spanning 6 days.

Blotting, Western↗
PubMed2001

Expression and purification of Dengue virus type 2 envelope protein as a fusion with hepatitis B surface antigen in Pichia pastoris.

The methylotrophic yeast, Pichia pastoris, has been used as a host to express the envelope protein (Den2E) of dengue type 2 virus (NGC strain) as a chimera with hepatitis B surface antigen (HBsAg): a protein known to self assemble into virus-like particles (VLPs) and to be efficiently expressed in P. pastoris. The Den2E gene used in this study is a truncated version encoding the first 395 amino acid (aa) residues of the mature Den2E protein; the HBsAg gene encodes the full length 226 aa HBsAg protein. Two in-frame gene fusions were constructed for intracellular expression in P. pastoris. The first one contains the HBsAg gene as the 5' partner and the Den2E gene as the 3'partner (HBsAg-Den2E). In the second one, the relative positions of the two partners of the gene fusion were reversed to create the hybrid Den2E-HBsAg gene. These fusion genes were integrated into the genome of P. pastoris under the control of the methanol-inducible alcohol oxidase (AOX1) promoter. Of the two fusions, the Den2E-HBsAg gene was expressed at higher levels in P. pastoris based on Northern analysis. The hybrid protein ( approximately 68 kDa) expressed by this clone was purified to near homogeneity using a combination of acid precipitation, hydrophobic interaction, and immunoaffinity chromatographic steps. Final purification achieved was approximately 1400-fold with a yield of approximately 26%. The chimeric protein was found to possess the ability to assemble into high molecular weight aggregates (akin to HBsAg particles). The recombinant fusion protein eluted close to the void volume of a Sepharose CL-4B column indicating its macromolecular nature. On a CsCl density gradient the recombinant fusion protein sedimented to a position very similar to that of HBsAg VLPs. The hybrid protein is recognized by the two neutralizing monoclonals against the two components of the chimeric protein.

Antibodies, Monoclonal↗
PubMed2001

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin.

Microbial reduction of alpha-chloroketone to alpha-chlorohydrin was studied as one of the approaches for construction of the chiral center of the corresponding epoxide. About 100 microorganisms covering many species of Candida, Pichia, Hansenula, Geotrichum, Rhodococcus and Aureobasidium were screened to reduce the alpha-chloroketone stereospecifically. Many strains provided the R-alpha-chlorohydrin with 100% enantiomeric excess (ee), e.g., Candida sonorensis SC 16117, Geotrichum candidum SC 5469, Rhodotorula glutinis SC 16293, Sphingomonas paucimobilis SC 16113, Pichia silvicola SC 16159 and Rhodococcus equi SC 15835. Few microorganisms showed preferential formation of S-alpha-chlorohydrin after reduction. Among them, Pichia pinus SC 13864 and two Pichia methanolica strains SC 16116 and SC 13860 were the best, providing the S-alpha-chlorohydrin with ee of 88%, 79% and 78%, respectively. The enantiospecificity of the reduction by these Pichia species can be modified by changing the pH or prior heat treatment of the cells and S-alpha-chlorohydrin with > or =95% ee was obtained by appropriate modification of reaction conditions.

Bacteria↗
PubMed2001

Role of TP53 in repair of N-(deoxyguanosin-8-yl)-4-aminobiphenyl adducts in human transitional cell carcinoma of the urinary bladder.

The global genomic repair of DNA adducts was examined in human papillary transitional cell carcinoma (TCC) cell lines after exposure to N:-hydroxy-4-acetylaminobiphenyl (N-OH-AABP), the proximate carcinogenic metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). (32)P-post-labeling analysis of TCC cultures exposed to N-OH-AABP revealed a major adduct, identified as the 3',5'-bisphosphate derivative of N-(deoxyguanosin-8-yl)-4-aminobiphenyl (dG-C8-ABP). The amount of adduct formation in TCC10 was dependent upon the dose and the duration of exposure and ranged between 1 and 5 adducts/10(7) nucleotides. To test if p53 regulates repair of the dG-C8-ABP adduct in genomic DNA, an isogeneic set of cell lines was obtained by infection of the TCC10 cultures with a retroviral construct expressing a trans-dominant mutant of p53, namely a Val-->Ala mutation at codon 143. The TDM143-TCC10 line expressing the mutant form of p53 was selected. The rate of repair of dG-C8-ABP was compared between TCC10 and TDM143-TCC10 cultures after treatment with 15 microM N-OH-AABP. The rate of disappearance of the adduct was monitored over a period of time after chemical treatment. (32)P-post-labeling analysis of dG-C8-ABP in parental TCC10 showed its rapid removal, the majority of adducts disappearing within 48 h. In contrast to TCC10, TDM143-TCC10 was relatively slower in removal of dG-C8-ABP. After 24 h DNA repair TDM143-TCC10 showed an approximately 3-fold greater amount of dG-C8-ABP compared with TCC10. These results imply that p53 plays a role in the repair of ABP adducts and that in p53 null cells the unrepaired DNA damage could cause accumulation of mutations, which might contribute to increased genomic instability and neoplastic progression.

Aminobiphenyl Compounds↗
PubMed2001

Crystallization and preliminary X-ray analysis of Borrelia burgdorferi outer surface protein C (OspC).

Single crystals of the outer surface protein C (OspC) from Borrelia burgdorferi HB19 have been obtained by the vapor-diffusion method. These crystals belong to space group P2(1), with unit-cell parameters a = 66.218, b = 46.113, c = 112.079 A, beta = 99.30 degrees, and diffract to at least 2.2 A resolution. Native data have been collected from flash-frozen crystals at the National Synchrotron facility of Brookhaven National Laboratory. There are two dimers per asymmetric unit, related by a non-crystallographic twofold axis and a pseudo-translational symmetry.

Amino Acid Sequence↗
PubMed2001

Epstein-Barr virus SM protein interacts with mRNA in vivo and mediates a gene-specific increase in cytoplasmic mRNA.

SM is an Epstein-Barr virus (EBV) gene expressed during early lytic replication of EBV. SM encodes a nuclear phosphoprotein that functions as a posttranscriptional regulator of gene expression. SM has been implicated in several aspects of gene regulation, including nuclear mRNA stabilization, posttranscriptional processing, and nuclear mRNA export. Activation by SM is promoter independent but gene specific. The mechanism by which SM selectively activates some EBV target genes or heterologous reporter genes remains to be determined. SM binds RNA in vitro, suggesting that sequence- or structure-specific mRNA interactions might mediate SM specificity. We have further analyzed RNA binding by SM and demonstrated that proteolytic cleavage of SM and consequent exposure of an arginine-rich region are necessary to allow RNA binding in vitro. However, SM mutants with deletions of this arginine-rich region localized normally in the nucleus and were fully functional in gene activation. We therefore developed an assay to study in vivo interactions of SM with target mRNAs based on immunoprecipitation of SM from cell lysates followed by RNase protection analysis. Using this assay, we demonstrated that SM forms complexes with specific mRNAs in vivo. SM binds mRNAs from both SM-responsive as well as nonresponsive intronless genes and increases the nuclear accumulation of both types of mRNAs. In addition, SM preferentially associates with newly transcribed mRNAs. These data indicate that SM forms complexes with mRNAs in the nucleus and enhances their nuclear accumulation. However, SM does not enhance cytoplasmic accumulation of all transcripts that it binds to the same degree, suggesting that additional mRNA-specific characteristics, such as nuclear retention motifs or binding sites for cellular proteins, also determine responsiveness to SM.

Cytoplasm↗
PubMed2001

Biochemical markers in the management of suspected acute myocardial infarction in the emergency department.

OBJECTIVES: To compare cardiac troponin T, myoglobin, CK, CKMB activity, CKMB mass and the initial electrocardiogram in the early diagnosis of myocardial infarction in the emergency department. Methods-Biochemical markers were measured at presentation in patients with a possible diagnosis of acute myocardial infarction. Based on the clinical notes, patients were grouped as "definite myocardial infarction" (n = 50), "definite no myocardial infarction" (n = 81) and "uncertain" (n = 96). Sensitivity and specificity and positive and negative predictive values were calculated using the 131 patients with definitely present or absent myocardial infarction. RESULTS: The initial electrocardiogram was more sensitive than any of the markers in the first six hours from symptom onset-sensitivity 74% (95%CI 61% to 88%). The positive predictive value of the initial electrocardiogram was 97% in the first six hours; the markers ranged from 47% to 67%. The negative predictive value of the initial electrocardiogram was 85% in the first six hours; the markers ranged from 61% to 70%. Four patients with non-diagnostic electrocardiograms presenting beyond six hours after pain onset had a myocardial infarct detected by at least three of the biochemical markers in each case. CONCLUSIONS: The electrocardiogram is of more diagnostic use than biochemical markers in the first six hours after the onset of pain, but biochemical markers give additional positive diagnostic information in patients presenting later than this. The negative predictive accuracy of biochemical markers is too low for a single sample to be useful for excluding myocardial infarction in the first six hours after onset of symptoms.

Creatine Kinase↗
PubMed2000

Biocatalytic preparation of a chiral synthon for a vasopeptidase inhibitor: enzymatic conversion of N(2)-

[4S-(4I,7I,10aJ)]1-Octahydro-5-oxo-4-[phenylmethoxy)carbonyl]amino]-7H-pyrido-[2,1-b] [1,3]thiazepine-7-carboxylic acid methyl ester (BMS-199541-01) is a key chiral intermediate for the synthesis of Omapatrilat (BMS-186716), a new vasopeptidease inhibitor under development. By using a selective enrichment culture technique we have isolated a strain of Sphingomonas paucimobilis SC 16113, which contains a novel L-lysine epsilon-aminotransferase. This enzyme catalyzed the oxidation of the epsilon-amino group of lysine in the dipeptide dimer N(2)-[N[phenyl-methoxy)-carbonyl] L-homocysteinyl] L-lysine)1,1-disulphide (BMS-201391-01) to produce BMS-199541-01. The aminotransferase reaction required alpha-ketoglutarate as the amino acceptor. Glutamate formed during this reaction was recycled back to alpha-ketoglutarate by glutamate oxidase from Streptomyces noursei SC 6007. Fermentation processes were developed for growth of S. paucimobilis SC 16113 and S. noursei SC 6007 for the production of L-lysine epsilon-amino transferase and glutamate oxidase, respectively. L-lysine epsilon-aminotransferase was purified to homogeneity and N-terminal and internal peptides sequences of the purified protein were determined. The mol wt of L-lysine epsilon-aminotransferase is 81 000 Da and subunit size is 40 000 Da. L-lysine epsilon-aminotransferase gene (lat gene) from S. paucimobilis SC 16113 was cloned and overexpressed in Escherichia coli. Glutamate oxidase was purified to homogeneity from S. noursei SC 6003. The mol wt of glutamate oxidase is 125 000 Da and subunit size is 60 000 Da. The glutamate oxiadase gene from S. noursei SC 6003 was cloned and expressed in Streptomyces lividans. The biotransformation process was developed for the conversion of BMS-201391-01 to BMS-199541-01 by using L-lysine epsilon-aminotransferase expressed in E. coli. In the biotransformation process, for conversion of BMS-201391-01 (CBZ protecting group) to BMS-199541-01, a reaction yield of 65-70 M% was obtained depending upon reaction conditions used in the process. Phenylacetyl or phenoxyacetyl protected analogues of BMS-201391-01 also served as substrates for L-lysine epsilon-aminotransferase giving reaction yields of 70 M% for the corresponding BMS-199541-01 analogs. Two other dipeptides N-[N[(phenylmethoxy)carbonyl]-L-methionyl]-L-lysine (BMS-203528) and N,2-[S-acetyl-N-[(phenylmethoxy)carbonyl]-L-homocysteinyl]-L-lysine (BMS-204556) were also substrates for L-lysine epsilon-aminotransferase. N-alpha-protected (CBZ or BOC)-L-lysine were also oxidized by L-lysine epsilon-aminotransferase.

Journal Article↗
PubMed2000

Synthesis of allysine ethylene acetal using phenylalanine dehydrogenase from Thermoactinomyces intermedius.

Allysine ethylene acetal [(S)-2-amino-5-(1,3-dioxolan-2-yl)-pentanoic acid (2)] was prepared from the corresponding keto acid by reductive amination using phenylalanine dehydrogenase (PDH) from Thermoactinomyces intermedius ATCC 33205. Glutamate, alanine, and leucine dehydrogenases, and PDH from Sporosarcina species (listed in order of increasing effectiveness) also gave the desired amino acid but were less effective. The reaction requires ammonia and NADH. NAD produced during the reaction was recyled to NADH by the oxidation of formate to CO(2) using formate dehydrogenase (FDH). PDH was produced by growth of T. intermedius ATCC 33205 or by growth of recombinant Escherichia coli or Pichia pastoris expressing the Thermoactinomyces enzyme. Using heat-dried T. intermedius as a source of PDH and heat-dried Candida boidinii SC13822 as a source of FDH,98%, but production of T. intermedius could not be scaled up. Using heat-dried recombinant E. coli as a source of PDH and heat-dried Candida boidinii 98%. In a third generation process, heat-dried methanol-grown P. pastoris expressing endogenous FDH and recombinant Thermoactinomyces98% ee.

Journal Article↗