Structure and expression of the Chlorobium vibrioforme hemA gene

The green sulfur bacterium, Chlorobium vibrioforme, synthesizes the tetrapyrrole precursor, -aminolevulinic acid (ALA), from glutamate via the RNA-dependent five-carbon pathway. A 1.9-kb clone of genomic DNA from C. vibrioforme that is capable of transforming a glutamyl-tRNA reductase-deficient, ALA-dependent, hemA mutant of Escherichia coli to prototrophy was sequenced. The transforming C. vibrioforme DNA has significant sequence similarity to the E. coli, Salmonella typhimurium, and Bacillus subtilis hemA genes and contains a 1245 base open reading frame that encodes a 415 amino acid polypeptide with a calculated molecular weight of 46174. This polypeptide has over 28% amino acid identity with the polypeptides deduced from the nucleic acid sequences of the E. coli, S. typhimurium, and B. subtilis hemA genes. No sequence similarity was detected, at either the nucleic acid or the peptide level, with the Rhodobacter capsulatus or Bradyrhizobium japonicum hemA genes, which encode ALA synthase, or with the S. typhimurium hemL gene, which encodes glutamate-1-semialdehyde aminotransferase. These results establish that hemA encodes glutamyl-tRNA reductase in species that use the five-carbon ALA biosynthetic pathway. A second region of the cloned DNA, located downstream from the hemA gene, has significant sequence similarity to the E. coli and B. subtilis hemC genes. This region contains a potential open reading frame that encodes a polypeptide that has high sequence identity to the deduced E. coli and B. subtilis HemC peptides. hemC encodes the tetrapyrrole biosynthetic enzyme, porphobilinogen deaminase, in these species. Preliminary evidence was obtained for the existence of a 3.0-kb polycistronic meassge that includes the hemA sequence, in exponentially growing C. vibrioforme cells. Results of condon usage analysis for the C. vibrioforme hemA gene indicate that green sulfur bacteria are more closely related to purple nonsulfur bacteria than to enteric bacteria. Sequences corresponding to a polyadenylation signal and a poly(A) attachment site were found immediately downstream from the 3 end of the hemA open reading frame.     

Engineering <Emphasis Type="Italic">Escherichia coli</Emphasis> for efficient coproduction of polyhydroxyalkanoates and 5-aminolevulinic acid

Single-cell biorefineries are an interesting strategy for using different components of feedstock to produce multiple high-value biochemicals. In this study, a strategy was applied to refine glucose and fatty acid to produce 5-aminolevulinic acid (ALA) and polyhydroxyalkanoates (PHAs). To express the ALA and PHAs dual-production system efficiently and stably, multiple copies of the poly-β-3-hydroxybutyrate (PHB) synthesis operon were integrated into the chromosome of Escherichia coli DH5αΔpoxB. The above strain harboring the ALA C5 synthesis pathway genes hemA and hemL resulted in coproduction of 38.2% PHB (cell dry weight, CDW) and 3.2 g/L extracellular ALA. To explore coproduction of ALA and poly(3-hydroxybutyrate-co-3-hydroxyvalerate) (PHBV), the PHBV synthetic pathway was also integrated into engineered E. coli and coexpressed with hemA and hemL; cells produced 38.9% PHBV (CDW) with 10.3 mol% 3HV fractions and 3.0 g/L ALA. The coproduction of ALA with PHB and PHBV can improve the utilization of carbon sources and maximize the value derived from the feedstock.     

Cloning of hemA from Rhizobium sp. NGR234 and symbiotic phenotype of a gene-directed mutant in diverse legume genera

Summary The hemA gene which encodes -aminolaevulinic acid synthase (ALAS), was cloned and characterized from the broad host-range Rhizobium strain NGR234. A cosmid, identified by hybridization with the cloned gene of R. meliloti and complementation of an R. meliloti hemA mutant, was subcloned to yield a 5.5 kb fragment containing the entire NGR234 gene. A physical-genetic map was made and the interposon was introduced into a single EcoRI site which bisects the gene. The mutated gene was homogenotized into NGR234 to generate a hemA mutant, with a view to evaluating the role of rhizobial bacteroid ALAS activity for a wide variety of legume symbioses. The mutant strain formed an ineffective (Fix^–) symbiosis with all tested host plants. These included tropical legumes that produce either indeterminate (Leucaena) or determinate (Desmodium, Macroptilium, Lablab, Vigna) root nodules.Abbreviations ALA -aminolaevulinic acid - ALAS aminolaevulinic acid synthase - Lb leghaemoglobin - Lb-haem haem moiety of leghaemoglobin     

Respiratory deficient mutants of Rhodopseudomonas capsulata

The facultative photosynthetic bacterium Rhodopseudomonas capsulata was mutagenized by transfer of the plasmid pSUP201::Tn5 from Escherichia coli to R. capsulata. Mutants defective in cytochrome oxidase and other respiratory functions were selected by replica plating, NADI-reaction and immunological methods. Among 20,000 mutants no clone was detected, which lacks the 65,000-protein of the cytochrome oxidase, but many mutants have been isolated which were cytochrome oxidase deficient (or inactive). Other mutants excrete heme and cytochrome c into the medium and lack cytochrome c ₂.Abbreviations Ap ampicillin - CIE crossed immunoelectrophoresis - cyt cytochrome - Cm chloramphenicol - Km kanamycin - SDS sodium dodecylsulfate - Tc tetracycline     

D-Amino acid dehydrogenase of Escherichia coli K12: positive selection of mutants defective in enzyme activity and localization of the structural gene

Summary A method for the positive selection of dadA mutants defective in Dolor-amino acid dehydrogenase has been devised. It consists in isolating mutants resistant to -chroro-Dolor-alanine and screening for mutant colony color on a special agar medium. All 70 Escherichia coli K12 dadA mutants isolated either by this method or by other selection procedures map at a locus which is near to hemA and closely linked with dadR. Since some of the dadA mutants are thermosensitive in Dolor-methionine utilization in vivo and have thermolabile Dolor-amino acid dehydrogenase in vitro, it is proposed that the dadA gene codes for the enzyme structure. The broad substrate specificity, apparent membrane localization, inducibility by alanine, and repressibility by glucose strongly suggest that the Dolor-amino acid dehydrogenase coded by the dadA gene is a species variant of the enzyme described under the same name in Salmonella typhimurium. It may be identical or homologous with the enzymes described under the names alaninase, Dolor-alanine oxidase or Dolor-alanine dehydrogenase in E. coli K12 or B.     

Biosynthesis of vitamin B12

Radioactivity from [1-¹⁴C]riboflavin was incorporated into the 5,6-dimethylbenzimidazole moiety of Vitamin B₁₂ in the aerobes Bacillus megaterium, Nocardia rugosa and Streptomyces sp. as well as in the aerotolerant anaerobe Propionibacterium freudenreichii, but not in the anaerobe Eubacterium limosum.As recently published for E. limosum, also in the anaerobe Clostridium barkeri radioactivity from [1-¹⁴C]glycine and [2-¹⁴C]glycine was found in the 5,6-dimethylbenzimidazole moiety, but not in the corrin moiety. The addition of l-[methyl-¹⁴C]methionine to C. barkeri led to the labeling of the corrin moiety and the 5,6-dimethylbenzimidazole moiety, showing that the seven extra methyl groups in the corrin ring as well as the two methyl groups of the base part originate from this precursor.In Clostridium thermoaceticum, forming the vitamin B₁₂ analog 5-methoxybenzimidazolylcobamide, [1-¹⁴C]glycine and [2-¹⁴C]glycine were also incorporated into the 5-methoxybenzimidazole moiety, but not into the corrin ring.In E. limosum l-[U-¹⁴C]glutamate led to the labeling of the corrin ring of vitamin B₁₂, but not of its base moiety.There results together with data from the literature indicate that a common biosynthetic pathway might exist for the corrinoid biosynthesis in aerobic microorganisms, and in those aerotolerant anaerobes like the Propionibacteria, which form the 5,6-dimethylbenzimidazole moiety of vitamin B₁₂ only under aerobic conditions. They also show that this pathway differs from the pathway found in anaerobic bacteria.     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

Genetic and physical characterization of putP, the proline carrier gene of Escherichia coli K12

Summary Two new mutants of E. coli K12, strains PT9 and PT32 were isolated, that were defective in proline transport. They had no high affinity proline transport activity, but their cytoplasmic membranes retained proline binding activity with altered sensitivity to inhibition by p-chloromercuribenzoate(pCMB). The lesion was mapped at the putP gene, which is located at min 23 on the revised E. coli genetic map (Bachmann 1983) as a composite gene in the proline utilization gene cluster, putP, putC, and putA, arranged in this order. The putC gene was shown to regulate the synthesis of proline dehydrogenase (putA gene product).Hybrid plasmids carrying the put region (Motojima et al. 1979; Wood et al. 1979) were used to construct the physical map of the put region. The possible location of the putP gene in the DNA segment was determined by subcloning the putP gene, genetic complementation, and recombination analyses using several proline transport mutants.Abbreviations pCMB p-chloromercuribenzoate - DM Davis and Mingioli - Ap ampicillin - NTG N-methyl-N-nitro-N-nitrosoguanidine - EMS ethylmethane sulfonate - Str streptomycin - Tet tetracycline - Ac l-azetidine-2-carboxylic acid - DHP 3, 4-dehydro-d,l-proline - MTT 3-(4,5-dimethyl-2)2,5-diphenyl tetrazolium bromide - Tris tris(hydroxymethyl)aminomethane - EDTA ethylenediamine tetraacetic acid - Kan kanamycin - Spc spectinomycin     

Genetic control of chlorophyll biosynthesis: Regulation of delta-aminolevulinate synthesis in Chlamydomonas

Summary A chlorophyll-deficient mutant, br _s -1, of Chlamydomonas reinhardtii has been shown to accumulate low levels of an intermediate, protoporphyrin (PROTO), and to form light-brown colonies. A double mutant, br _s -1 r-1, accumulates 15-fold more PROTO than br _s -1 and forms dark-brown colonies. Enzymes synthesizing the first intermediate of chlorophyll, delta-aminolevulinate (ALA), from these two mutants and the wild-type are equally sensitive to inhibition by heme. The activity of ALA-synthesizing enzymes from br _s -1 r-1 is similar to that of the wild-type and is more than threefold that of br _s -1. It is proposed that the ALA-synthesizing enzymes in br _s -1 are under repression while r-1 is a mutation of the regulatory gene and consequently derepresses the synthesis of its own ALA-synthesizing enzymes. In addition, by mutagenizing br _s -1, we isolated six more double mutants having the same phenotype as br _s -1 r-1. Five of them are identical to br _s -1 r-1, the remaining one (db-10) carries a second mutation nonallelic to r-1. The ALA-synthesizing enzymes from db-10 are much less sensitive to heme inhibition than those from the wild type. It is proposed that ALA synthesis in Clamydomonas is regulated both allosterically and genetically.Abbreviations PROTO protoporphyrin - ALA delta-aminolevulinate - Mg-PROTO magnesium-protoporphyrin - GSA glutamate-1-semialdehyde     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Analogue-resistant and auxotrophic mutants ofMethanobacterium thermoautotrophicum

The death rate ofMethanobacterium thermoautotrophicum strain Marburg upon exposure toN-methyl-N-nitro-N-nitrosoguanidine under anaerobic conditions was of the same order of magnitude as the death rates that have been reported forEscherichia coli. Cultures of the methanogenic bacterium, mutagenized by nitrosoguanidine-treatment and grown under non-selective conditions, yielded mutants resistant toDL-ethionine (30 mM) or to 2-bromoethane sulfonic acid (3.8 mM). No mutants were observed in untreated controls. Among 1500 clones obtained from nitrosoguanidine-treated cell suspensions there were 6 mutants requiring a single growth factor each, namelyl-leucine,l-phenylalanine, thiamine (2 mutants) or adenosine (2 mutants). Three mutant-strains were studied in more detail. They were genetically stable (no revertants among 10⁹ cells), and wild type growth rates were restored by 5 mml-leucine, 0.4 mM adenosine and 0.03 mM thiamine, respectively.Abbreviations 2-BES 2-bromoethanesulfonic acid - MIC minimum inhibitory concentration     

Stimulation of the anaerobic growth ofSalmonella typhimurium by reduction ofl-carnitine,carnitine derivatives and structure-related trimethylammonium compounds

In view of the development of al-carnitine deficiency, the metabolism ofl-carnitine and structure-related trimethylammonium compounds was studied inSalmonella typhimurium LT₂ by means of thin-layer chromatography (TLC).l-Carnitine, crotonobetaine and acetyl-l-carnitine stimulated the anaerobic growth in a complex medium significantly. The stimulation depended on the formation of -butyrobetaine. The reduction ofl-carnitine proceeded in two steps: (1) Dehydration of thel-carnitine to crotonobetaine, (2) hydrogenation of crotonobetaine to -butyrobetaine. The reduction of crotonobetaine was responsible for the growth stimulation. Terminal electron acceptors of the anaerobic respiration such as nitrate and trimethylamine N-oxide, but not fumarate, suppressed the catabolism ofl-carnitine completely. Glucose fermentation, too, inhibited the reduction ofl-carnitine but optimal growth with a high carnitine catabolism was achieved byd-ribose. The esters of carnitine with medium- and long-chain fatty acids inhibited the growth considerably because of their detergent properties.Abbreviations TLC thin-layer chromatography     

Fate of the porphyrin cofactors during the light-dependent turnover of catalase and of the photosystem II reaction-center protein D1 in mature rye leaves

The apoprotein of the enzyme catalase (EC 1.11.1.6) was shown to exhibit a light-dependent turnover in leaves. Present results indicate that photoinactivation of the enzyme was not accompanied by a synchronous destruction and new synthesis of its heme moiety. In rye (Secale cereale L.) leaves the catalase content was not depleted in light when porphyrin synthesis was inhibited by gabaculine. Photoinactivation of purified bovine liver or rye leaf catalase in vitro was not accompanied by concomitant damage to the heme groups. Both the incorporation of -[³H]aminolevulinic acid ([³H]ALA) into catalase-heme and its apparent turnover increased with irradiance. However, the apparent half-life of the catalase-heme was much longer than that of its apoprotein. It is probable that not only degradation but also an exchange with the free heme pool contributed to the apparent turnover of radioactivity of the catalase-heme. Part of the chlorophyll (Chl) associated with photosystem II (PS II) had a preferential light-induced turnover, and repair of PS II appeared to require new Chl synthesis also in mature green rye leaves. The activity of PS II, indicated by the ratio of variable to maximal fluorescence (F_v/F_m), rapidly declined in the presence of gabaculine in light and the reaction-center proteins D1 and D2 were depleted. When segments of mature green rye leaves were labeled with [³H]ALA and incorporation into Chl-protein complexes analysed after electrophoretic separation in the presence of Deriphat, the highest radioactivity was observed in the core complex of PS II, while PS I and the light-harvesting complex of PS II (LHC II) were unlabeled. In greening etiolated leaves highest incorporation was observed in LHC II. Both the incorporation of [³H]ALA into the PS II core complex of green rye leaves and its turnover increased with irradiance. However, the apparent half-life of the PS II-bound labeled porphyrin compounds (mainly Chl) was considerably longer than that of the reaction-center protein D1 under identical conditions.Abbreviations ALA -aminolevulinic acid - CII Core complex of PS II - Chl chlorophyll - DMSO dimethyl sulfoxide - F_v/F_m ratio of variable to maximal chlorophyll fluorescence - LHC light-harvesting complex - PAR photosynthetically active radiation We thank the Deutsche Forschungsgemeinschaft for financial support. Technical assistence by B. Kramer and Ch. van Oijen is greatly appreciated. We are grateful to Dr. Johanningmeier and Dr. Godde (Lehrstuhl für Biochemie der Pflanzen, Universität Bochum, Germany) for providing antisera against the D1 and D2 proteins and Dr. M. Schmidt (Botanisches Institut, Universität Frankfurt am Main, Germany) for valuable advice. Deriphat 160 was kindly supplied by Henkel Corp., Hoboken, N.J., USA.     

Isolation and classification of chlorophyll-deficient xantha mutants of Arabidopsis thaliana

Mutant lines of Arabidopsis thaliana that are either blocked at various steps of the biosynthetic pathway of chlorophyll (Chl) or that are disturbed in one of the subsequent steps leading to the assembly of an active photosynthetic membrane were isolated by screening for Chl-deficient xantha (xan) mutants. Only mutants that segregated in a 31 ratio, that contained the same carotenoid spectrum as etiolated wild-type seedlings and less than 2% of the Chl of wild-type control seedlings, and whose Chl content was not affected by the addition of sucrose to the growth medium were selected for a more detailed analysis. As a final test for the classification of the selected mutants, light-grown xan mutants were vacuum-infiltrated and incubated with the common precursor of tetrapyrroles, -aminolevulinic acid (ALA), in the dark. Two major groups of mutants could be distinguished. Some of the mutants were blocked at various steps of the Chl pathway between ALA and protochlorophyllide (Pchlide) and did not accumulate the latter in the dark. The other mutants accumulated Pchlide in the dark regardless of whether exogenous ALA was added. This latter group could be subdivided into mutants with a biochemical lesion in a recently discovered second light-dependent Pchlide reduction step that occurs in green plants and mutants that have blocks in the assembly of Chl protein complexes. In the present work a total of seven different loci could be defined genetically in Arabidopsis that affect the synthesis of Chl and its integration into the growing photosynthetic membrane.Abbreviations ALA -aminolevulinic acid - Chl chlorophyll - Chlide chlorophyllide - Pchlide protochlorophyllide - POR NADPH-Protochlorophyllide oxidoreductase - xan xantha This study was initiated while one of the authors (K.A.) was on sabbatical leave in the laboratory of Dr. C. Somerville (MSU, East Lansing, Mich., USA). We are extremely grateful to Dr. Somerville and his coworkers for advice and support during this time. This research was supported by the Deutsche Forschungsgemeinschaft and the Schweizerischer Nationalfonds.     

Defects in cytochrome cd 1-dependent nitrite respiration of transposon Tn5-induced mutants from Pseudomonas stutzeri

Mutants with defective respiratory nitrite utilization (Nir^- phenotype) were obtained by transposon Tn5 insertion into genomic DNA of the ZoBell strain of Pseudomonas stutzeri. Three representative mutants were characterized with respect to their activities of nitrite and nitric oxide reduction, cytochrome cd ₁ content, and pattern of soluble c-type cytochromes. Mutant strain MK201 over-produced cytochrome c ₅₅₂ about fourfold by comparison with the wild type, but possessed an in vitro functional cytochrome cd ₁. Mutant strain MK202 lacked cytochrome cd ₁ and, simultaneously, had low amounts of cytochrome c ₅₅₂ and the split -peak c-type cytochrome. Strain MK203 synthesized nitrite reductase defective in the heme d ₁ prosthetic group. Irrespective of these biochemically distinct Nir^- phenotypes, all mutants preserved the nitric oxidereducing capability of the wild type. The mutant characteristics demonstrate that cytochrome cd ₁ is essential for nitrite respiration of P. stutzeri and establish the presence of a nitric oxide-reducing system distinct from cytochrome cd ₁. They also indicate the functional or regulatory interdependence of c-type cytochromes.     

N-acetylglucosaminyltransferase substrates prepared from glycoproteins by hydrazinolysis of the asparagine-N-acetylglucosamine linkage. Purification and structural determination of oligosaccharides with mannose andN-acetylglucosamine at the non-reducing termini

Sixteen asparagine-linked oligosaccharides ranging in size from (Man)₂(GlcNAc)₂ (Fuc)₁ to (GlcNAc)₆(Man)₃(GlcNAc)₂ were obtained from human ₁-acid glycoprotein and fibrinogen, hen ovomucoid and ovalbumin, and bovine fetuin, fibrin and thyroglobulin by hydrazinolysis, mild acid hydrolysis and glycosidase treatment. The oligosaccharides hadN-acetylglucosamine at the reducing termini and mannose andN-acetylglucosamine residues at the non-reducing termini and were prepared for use asN-acetylglucosaminyltransferase substrates. Purification of the oligosaccharides involved gel filtration and high performance liquid chromatography on reverse phase and amine-bonded silica columns. Structures were determined by 360 MHz and 500 MHz proton nuclear magnetic resonance spectroscopy, fast atom bombardment-mass spectrometry and methylation analysis. Several of these oligosaccharides have not previously been well characterized.Abbreviations bis bisecting GlcNAc - DMSO dimethylsulfoxide - FAB fast atom bombardment - Fuc l-fucose - Gal d-galactose - GLC gas-liquid chromatography - GlcNAc or Gn N-acetyl-d-glucosamine - HPLC high performance liquid chromatography - Man or M d-mannose - MES 2-(N-morpholino)ethanesulfonate - MS mass spectrometry - NMR nuclear magnetic resonance - PIPES piperazine-N,N-bis(2-ethane sulfonic acid) the nomenclature of the oligosaccharides is shown in Table 1.     

Symbiotically defective histidine auxotrophs of Bradyrhizobium japonicum

Four histidine auxotrophs of Bradyrhizobium japonicum strain USDA 122 were isolated by random transposon Tn5 mutagenesis. These mutants arose from different, single transposition events as shown by the comparison of EcoRI and XhoI-generated Tn5 flanking sequences of genomic DNA. The mutants grew on minimal medium supplemented with l-histidine or l-histidinol but failed to grow with l-histidinol phosphate. While two of the muants were symbiotically defective and did not form nodules on Glycine max cvs. Lee and Peking and on Glycine soja, the other two mutants were symbiotically competent. Reversion to prototrophy occurred at a frequency of about 10^-7 on growth medium without added antibiotics, but prototrophs could not be isolated from growth medium containing 200 g/ml kanamycin and streptomycin. The prototrophic revertants formed nodules on all the soybean cultivars examined. When histidine was supplied to the plant growth medium, both nodulation deficient mutants formed effective symbioses. On histidine unamended plants, nodules were observed infrequently. Three classes of bacterial colonies were isolated from such infrequent nodules: class 1 were kanamycin resistant-auxotrophs; class 2 were kanamycin sensitive-prototrophs; and class 3 were kanamycin-sensitive auxotrophs. Our results suggest that two Tn5 insertion mutations in B. japonicum leading to histidine auxotrophy, affect nodulation in some way. These mutations are in regions that show no homology to the Rhizobium meliloti common nodulation genes.     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase