Genetic and cytological analyses of a partial-female-sterile mutant (PS-1) in soybean (Glycine max; Leguminosae)

Soybean partial-female-sterile mutant 1 (PS-1) was recovered from a gene-tagging study. The objectives were to study the inheritance, linkage, allelism, and certain aspects of the reproductive biology of the PS-1 mutant. For inheritance and linkage tests, PS-1 was crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient mutant CD-1, also recovered from the gene-tagging study. For allelism tests, reciprocal crosses were made with PS-1 and three other partial-sterile mutants (PS-2, PS-3, and PS-4) recovered from the same gene-tagging study. The PS-1 mutant is inherited in a 3:1 ratio and is a single recessive gene. Linkage results indicated that the gene for partial sterility in PS-1 is not linked either to the w4 locus or to the CD-1 locus. Allelism tests showed that the gene in PS-1 is nonallelic to the gene in PS-2, PS-3, and PS-4. Investigations of developing and mature pollen indicated no differences in morphology, stainability, or fluorescence between normal and partial-sterile genotypes. The PS-1 mutant is completely male fertile. Confocal scanning laser microscopy was used to determine that early embryo abortion in PS-1 is due indirectly to abnormal migration of the fused polar nucleus, which prevented it from being fertilized. Subsequent absence of endosperm development leads directly to abortion of the proembryo.     

Stimulation of clover root hair infection by lectin-binding oligosaccharides from the capsular and extracellular polysaccharides of Rhizobium trifolii

A polysaccharide depolymerase isolated from the phage lysate of Rhizobium trifolii 4S was used to fragment capsular polysaccharides (CPS) and extracellular polysaccharides (EPS) of R. trifolii 0403 into oligosaccharides. These products were analyzed for clover lectin (trifoliin A)-binding ability, effect on infection of white clover root hairs, and changes in glycosyl and noncarbohydrate composition with culture age. The oligosaccharides from CPS of cultures grown on agar plates for 3, 5, and 7 days exhibited lectin-binding ability at levels similar to those of the corresponding intact CPS. The intact EPS did not bind to clover lectin, although the oligosaccharide fragments from EPS did. In contrast, oligosaccharides from deacetylated CPS had less than half the lectin-binding ability of the native polysaccharide substrate. The CPS from 5-day-old cultures, its corresponding oligosaccharide fragments, and the oligosaccharide fragments of EPS from 5-day-old cultures, all at a concentration of 2.5 micrograms per seedling, stimulated infection thread formation in root hairs of clover seedlings inoculated with R. trifolii 0403. Thus, this bacteriophage-induced polysaccharide depolymerase converted the acidic CPS and EPS of R. trifolii 0403 into biologically active oligosaccharides capable of binding trifoliin A and stimulating root hair infection. The amount of the noncarbohydrate substitutions (pyruvate, acetate, and ether-linked 3-hydroxybutyrate) in the CPS oligosaccharides changed with culture age as shown by 1H-nuclear magnetic resonance spectroscopy. The binding of trifoliin A, therefore, appears to be sensitive to changes in the degree of substitution of noncarbohydrate substitutions in the CPS of R. trifolii 0403.     

Exopolysaccharide Colanic Acid and Its Occurrence in the Enterobacteriaceae

A study of strains from the genera Salmonella, Escherichia, and Aerobacter has shown that under appropriate conditions many strains produce an exopolysaccharide slime of identical composition, which has been identified as colanic acid on the basis of its chemical composition and its sensitivity to certain bacteriophage-induced depolymerase enzymes. Chemical analysis shows that the polysaccharide contains O-acetyl groups in addition to the sugars glucose, galactose, fucose, and glucuronic acid. Mild acid hydrolysis has led to the isolation of a β-glucosylfucose in addition to glucuronic acid containing oligosaccharides. Many strains were found to synthesize colanic acid under normal conditions of growth or under conditions favoring polysaccharide synthesis, whereas others only synthesized colanic acid when the control mechanism was derepressed by p-fluorophenylalanine.     

Primary structure of the 2-O-methyl-alpha-L-fucose-containing side chain of the pectic polysaccharide,rhamnogalacturonan II

A 2-O-methylfucosyl-containing heptasaccharide was released from red wine rhamnogalacturonan II (RG-II) by acid hydrolysis of the glycosidic linkage of the aceryl acid residue (AceA) and purified to homogeneity by size-exclusion and high-performance anion-exchange chromatographies. The primary structure of the heptasaccharide was determined by glycosyl-residue and glycosyl-linkage composition analyses, ESIMS, and by 1H and 13C NMR spectroscopy. The NMR data indicated that the pyranose ring of the 2,3-linked L-arabinosyl residue is conformationally flexible. The L-Arap residue was confirmed to be alpha-linked by NMR analysis of a tetraglycosyl-glycerol fragment, [alpha-L-Arap-(1-->4)-beta-D-Galp-(1-->2)-alpha-L-AcefA-(1-->3)-beta-L-Rhap-(1-->3)-Gro], generated by Smith degradation of RG-II. Our data together with the results of a previous study,(1) establish that the 2-O-Me Fuc-containing nonasaccharide side chain of wine RG-II has the structure (Api [triple bond] apiose): [see structure]. Data are presented to show that in Arabidopsis RG-II the predominant 2-O-MeFuc-containing side chain is a mono-O-acetylated heptasaccharide that lacks the non-reducing terminal beta-L-Araf and the alpha-L-Rhap residue attached to the O-3 of Arap, both of which are present on the wine nonasaccharide.     

Isolation and characterization of a novel acidic polysaccharide containing tartrate and glyoxylate residues from the mineralized scales of a unicellular coccolithophorid alga Pleurochrysis carterae.

The characterization of mineral-associated polyanions from the unicellular alga Pleurochrysis carterae is described. This species is useful for the study of mineralization, because it produces calcified scales known as coccoliths in homogeneous cell culture. Three acidic polysaccharides (PS-1, PS-2, and PS-3) were extracted from the coccoliths with EDTA and were separated and purified by differential precipitation with magnesium ions and chromatography on DEAE-cellulose. PS-1 and PS-3 are predominantly polymers of galacturonic acid containing lesser amounts of other monosaccharides. PS-2 has an unusual structure. Chemical, enzymatic, and two-dimensional NMR analyses demonstrate that the repeating unit of PS-2 is [----4)D-glucuronate(beta 1----2)meso-tartrate(3----1)glyoxylate(1-]n. Thus PS-2 has a high density of negatively charged groups available for calcium ion binding, similar to the phosphoprotein polyanions of other species. Polysaccharides containing tartrate and/or glyoxylate have not been previously described; these residues may be introduced into PS-2 by a postpolymerization process involving oxidative cleavage of glucuronate or mannuronate residues.     

Enzymatic hydrolysis of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4

Enzymatic degradability has been investigated for a series of bacterial poly(3-hydroxybutyrate-co-3-hydroxypropionate)s (P(3HB-co-3HP)s) with 3-hydroxypropionate (3HP) unit contents from 11 to 86 mol % as well as poly(3-hydroxybutyrate) (P(3HB)) and chemosynthesized poly(3-hydroxypropionate) (P(3HP)). The behavior of degradation by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pikettii T1 and Acidovorax Sp. TP4, defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were compared in relation to the thermal properties and crystalline structures of the PHA samples elucidated by differential scanning calorimetry and wide-angle X-ray diffraction. The degradation products were characterized by high-performance liquid chromatography and one- (1D) and two-dimension (2D) (1)H NMR spectroscopy. It was found that the PHA depolymerase of Acidovorax Sp. TP4 showed degradation behavior different from that shown by depolymerase of R. pikettii T1. PHA depolymerase from Acidovorax Sp. TP4 degraded the P(3HB-co-3HP) films with lower crystallinity in higher rates than those with higher crystallinity, no matter what kinds of crystalline structures they formed. In contrast, PHA depolymerase from R. pikettii T1 degraded P(3HB-co-3HP) films forming P(3HB) crystalline structure in higher rates than those forming P(3HP)s. The increase in amorphous nature of the P(3HB-co-3HP) films with P(3HB)-homopolymer-like crystalline structure increases and then decreases the rate of degradation by depolymerase from R. pikettii T1. The 3-hydroxybutyrate (3HB) monomer was produced as a major product by the hydrolysis of P(3HB) film by PHA depolymerase from Acidovorax Sp. TP4. The P(3HB-co-3HP) films could be degraded into 3HB and 3-hydroxypropionate (3HP) monomer at last, indicating that the catalytic domain of the enzyme recognized at least two monomeric units as substrates. While the PHA depolymerase from R. pikettii T1 hydrolyzed P(3HB) film into 3HB dimer as a major product, and the catalytic domain recognized at least three monomeric units. The degradation behavior of P(3HB-co-3HP) films by the PHA depolymerase of Acidovorax Sp. TP4 could be distinguished from that by the depolymerase of R. pikettii T1.     

Poly(3-hydroxyvalerate) depolymerase of Pseudomonas lemoignei

Pseudomonas lemoignei is equipped with at least five polyhydroxyalkanoate (PHA) depolymerase structural genes (phaZ1 to phaZ5) which enable the bacterium to utilize extracellular poly(3-hydroxybutyrate) (PHB), poly(3-hydroxyvalerate) (PHV), and related polyesters consisting of short-chain-length hxdroxyalkanoates (PHA(SCL)) as the sole sources of carbon and energy. Four genes (phaZ1, phaZ2, phaZ3, and phaZ5) encode PHB depolymerases C, B, D, and A, respectively. It was speculated that the remaining gene, phaZ4, encodes the PHV depolymerase (D. Jendrossek, A. Frisse, A. Behrends, M. Andermann, H. D. Kratzin, T. Stanislawski, and H. G. Schlegel, J. Bacteriol. 177:596-607, 1995). However, in this study, we show that phaZ4 codes for another PHB depolymeraes (i) by disagreement of 5 out of 41 amino acids that had been determined by Edman degradation of the PHV depolymerase and of four endoproteinase GluC-generated internal peptides with the DNA-deduced sequence of phaZ4, (ii) by the lack of immunological reaction of purified recombinant PhaZ4 with PHV depolymerase-specific antibodies, and (iii) by the low activity of the PhaZ4 depolymerase with PHV as a substrate. The true PHV depolymerase-encoding structural gene, phaZ6, was identified by screening a genomic library of P. lemoignei in Escherichia coli for clearing zone formation on PHV agar. The DNA sequence of phaZ6 contained all 41 amino acids of the GluC-generated peptide fragments of the PHV depolymerase. PhaZ6 was expressed and purified from recombinant E. coli and showed immunological identity to the wild-type PHV depolymerase and had high specific activities with PHB and PHV as substrates. To our knowledge, this is the first report on a PHA(SCL) depolymerase gene that is expressed during growth on PHV or odd-numbered carbon sources and that encodes a protein with high PHV depolymerase activity. Amino acid analysis revealed that PhaZ6 (relative molecular mass [M(r)], 43,610 Da) resembles precursors of other extracellular PHA(SCL) depolymerases (28 to 50% identical amino acids). The mature protein (M(r), 41,048) is composed of (i) a large catalytic domain including a catalytic triad of S(136), D(211), and H(269) similar to serine hydrolases; (ii) a linker region highly enriched in threonine residues and other amino acids with hydroxylated or small side chains (Thr-rich region); and (iii) a C-terminal domain similar in sequence to the substrate-binding domain of PHA(SCL) depolymerases. Differences in the codon usage of phaZ6 for some codons from the average codon usage of P. lemoignei indicated that phaZ6 might be derived from other organisms by gene transfer. Multialignment of separate domains of bacterial PHA(SCL) depolymerases suggested that not only complete depolymerase genes but also individual domains might have been exchanged between bacteria during evolution of PHA(SCL) depolymerases.     

The "intracellular" poly(3-hydroxybutyrate) (PHB) depolymerase of Rhodospirillum rubrum is a periplasm-located protein with specificity for native PHB and with structural similarity to extracellular PHB depolymerases

Rhodospirillum rubrum possesses a putative intracellular poly(3-hydroxybutyrate) (PHB) depolymerase system consisting of a soluble PHB depolymerase, a heat-stable activator, and a 3-hydroxybutyrate dimer hydrolase (J. M. Merrick and M. Doudoroff, J. Bacteriol. 88:60-71, 1964). In this study we reinvestigated the soluble R. rubrum PHB depolymerase (PhaZ1). It turned out that PhaZ1 is a novel type of PHB depolymerase with unique properties. Purified PhaZ1 was specific for amorphous short-chain-length polyhydroxyalkanoates (PHA) such as native PHB, artificial PHB, and oligomer esters of (R)-3-hydroxybutyrate with 3 or more 3-hydroxybutyrate units. Atactic PHB, (S)-3-hydroxybutyrate oligomers, medium-chain-length PHA, and lipase substrates (triolein, tributyrin) were not hydrolyzed. The PHB depolymerase structural gene (phaZ1) was cloned. Its deduced amino acid sequence (37,704 Da) had no significant similarity to those of intracellular PHB depolymerases of Wautersia eutropha or of other PHB-accumulating bacteria. PhaZ1 was found to have strong amino acid homology with type-II catalytic domains of extracellular PHB depolymerases, and Ser(42), Asp(138), and His(178) were identified as catalytic-triad amino acids, with Ser(42) as the putative active site. Surprisingly, the first 23 amino acids of the PHB depolymerase previously assumed to be intracellular revealed features of classical signal peptides, and Edman sequencing of purified PhaZ1 confirmed the functionality of the predicted cleavage site. Extracellular PHB depolymerase activity was absent, and analysis of cell fractions unequivocally showed that PhaZ1 is a periplasm-located enzyme. The previously assumed intracellular activator/depolymerase system is unlikely to have a physiological function in PHB mobilization in vivo. A second gene, encoding the putative true intracellular PHB depolymerase (PhaZ2), was identified in the genome sequence of R. rubrum.     

Genetic and cytological analyses of three lethal ovule mutants in soybean (Glycine max; Leguminosae).

Soybean partially sterile mutants 2, 3, and 4 (PS-2, PS-3, and PS-4), recovered from a gene-tagging experiment, were studied to clarify their inheritance, linkage, allelism, and reproductive biology. The PS-2, PS-3, and PS-4 mutants were maintained as heterozygotes and upon self-pollination segregated l fertile: l partially-sterile. For inheritance and linkage tests, all three PS mutants were crossed to flower color mutant Harosoy-w4 and to chlorophyll-deficient (CD) mutants CD-1 and CD-5, also recovered from the tagging study. For allelism tests, reciprocal crosses were made among the three partially sterile mutants. Linkage results indicated that the gene for partial sterility in the PS-2, PS-3, and PS-4 mutants was not linked either to the w4 locus or to the genes for chlorophyll deficiency. Studies of pollen development, pollen viability, and pollen-tube germination indicated no difference between normal and partially sterile genotypes. Linkage and allelism tests indicated that the gene in the three partially sterile mutants was not transmitted through the female when they were used as a female parent. A study of megagametogenesis indicated that the ovules from partially sterile plants had normal embryo sac development. Ovule abortion was due to failure of fertilization.     

Independence from the substrate of multiplication in nonclonal and clonal tumor cell populations

Experiments were made with sarcoma PS-103 cells cultured in vitro. The sarcoma was induced by implantation of a plastic plate into CBA mice. Clonal analysis of the cell culture demonstrated that 1) all 3 clones isolated from substrate (SB) grew in 1.2% methyl cellulose (MC) at the same efficiency as parental cells; 2) all 5 clones isolated from MC formed in a semi-solid medium 10-100-fold more colonies than PS-103. During the subcloning of one of PS-103 clones in solid substrate and in MC, it turned out that the majority of MC and SB subclones had the plating efficiency in MC similar to that in PS-103. Apparently, the PS-103 population contains clones with different degrees of anchorage independence.     

Enzymatic hydrolysis of chemosynthesized atactic poly(3-hydroxybutyrate) by poly(3-hydroxyalkanoate) depolymerase from Acidovorax Sp. TP4 and Ralstonia pickettii T1

The enzymatic degradability of chemosynthesized atactic poly([R,S]-3-hydroxybutyrate) [a-P(3HB)] by two types of extracellular poly(3-hydroxyalkanoate) (PHA) depolymerases purified from Ralstonia pickettii T1 (PhaZ(ral)) and Acidovorax Sp. TP4 (PhaZ(aci)), defined respectively as PHA depolymerase types I and II according to the position of the lipase box in the catalytic domain, were studied. The enzymatic degradation of a-P(3HB) by PhaZ(aci) depolymerase was confirmed from the results of weight loss and the scanning electron micrographs. The degradation products were characterized by one- and two-dimension (1)H NMR spectroscopy. It was found that a-P(3HB) could be degraded into monomer, dimer, and trimer by PhaZ(aci) depolymerase at temperatures ranging from 4 to 20 degrees C, while a-P(3HB) could hardly be hydrolyzed by PhaZ(ral) depolymerase in the same temperature range. These results suggested that the chemosynthesized a-P(3HB) could be degraded in the pure state by natural PHA depolymerase.     

Structure of the extracellular gelling polysaccharide produced by Enterobacter (NCIB 11870) species

The gelling polysaccharide produced by a species of Enterobacter (NCIB 11870) contains L-fucose, D-glucose, and D-glucuronic acid in the ratios 1:2:1. Analysis of the methylated and methylated, carboxyl-reduced polysaccharide revealed terminal non-reducing glucose, (1----3)-linked fucose, (1----3,1----4)-linked glucose, and (1----4)-linked glucuronic acid in the ratios 1:1:1.2:0.8. From the results of Smith degradation of the polysaccharide and spectroscopic studies of the acidic tetra- and octa-saccharides produced by bacteriophage-induced enzymic depolymerization of the polysaccharide, the following tetrasaccharide repeating-unit is proposed. (Formula: see text). This repeating-unit is identical to that of the capsular polysaccharide produced by Klebsiella aerogenes serotype K54 except for the absence of O-acetyl groups. The effects of the O-acetyl groups on the secondary structure and rheological properties of these polysaccharides are discussed.     

Identification of a novel PSD-95/Dlg/ZO-1 (PDZ)-like protein interacting with the C terminus of presenilin-1.

Presenilin-1 (PS-1) is the most causative Alzheimer gene product, and its function is not well understood. In an attempt to elucidate the function of PS-1, we screened a human brain cDNA library for PS-1-interacting proteins using the yeast two-hybrid system and isolated a novel protein containing a PSD-95/Dlg/ZO-1 (PDZ)-like domain. This novel PS-1-associated protein (PSAP) shares a significant similarity with a Caenorhabditis elegans protein of unknown function. Northern blot analysis revealed that PSAP is predominantly expressed in the brain. Deletion of the first four C-terminal amino acid residues of PS-1, which contain the PDZ domain-binding motif (Gln-Phe-Tyr-Ile), reduced the binding activity of PS-1 toward PSAP 4-fold. These data suggest that PS-1 may associate with a PDZ-like domain-containing protein in vivo and thus may participate in receptor or channel clustering and intracellular signaling events in the brain.     

The beta 1----2-D-xylose and alpha 1----3-L-fucose substituted N-linked oligosaccharides from Erythrina cristagalli lectin. Isolation, characterisation and comparison with other legume lectins

The carbohydrate moieties of Erythrina cristagalli lectin were released as oligosaccharides by hydrazinolysis, followed by N-acetylation and reduction with NaB3H4. Fractionation of the tritium-labelled oligosaccharide mixture by Bio-Gel P-4 column chromatography and high-voltage borate electrophoresis revealed that it is composed of five neutral oligosaccharides. Structural studies by sequential exoglycosidase digestion in combination with methylation analysis and two-dimensional 1H-NMR showed that the major component was the fucose-containing heptasaccharide Man alpha 3(Man alpha 6)(Xyl beta 2)Man beta 4GlcNAc beta 4(Fuc alpha 3)GlcNAcol. This is the first report of such a structure in plant lectins. Small amounts of the corresponding afucosyl hexasaccharide were also identified, as well as three other minor components. The structure of the heptasaccharide shows the twin characteristics of a newly established family of N-linked glycans, found to date only in plants. The characteristics are substitution of the common pentasaccharide core [Man alpha 3(Man alpha 6)Man beta 4GlcNAc beta 4GlcNAc] by a D-xylose residue linked beta 1----2 to the beta-mannosyl residue and an L-fucose residue linked alpha 1----3 to the reducing terminal N-acetylglucosamine residue. The oligosaccharide heterogeneity pattern for Erythrina cristagalli lectin was also found for the lectins from four other Erythrina species and the lectins of two other legumes, Sophora japonica and Lonchocarpus capassa.     

Molecular mapping of four ovule lethal mutants in soybean

We report genetic mapping of four soybean ovule lethal mutants, PS-1, PS-2, PS-3, and PS-4, which had been identified as female partial-sterile mutants from a gene-tagging study. The four mutants had been classified into two mutation classes: (1) PS-1—sporophytic mutation affects sporophytically expressed genes; and (2) PS-2, PS-3, and PS-4 mutants—female gametophyte-specific mutations affect gametophytically expressed genes and are transmitted through the male, but not the female gametes. Molecular mapping demonstrated that these four mutant genes and previously reported female-partial sterile gene, Fsp1, are located independently on soybean molecular linkage groups (MLG-) using SSR markers. PS-1, designated as Fsp2 and Genetic Type Collection number T364, is located between SSR markers Satt170 and Satt363 on MLG-C2 and linked by 13.9 cM and 12.1 cM, respectively. PS-2, designated as Fsp3 and Genetic Type Collection number T365H, is located between SSR markers Satt538 and Satt429 on MLG-A2 and linked by 13.3 cM and 25.4 cM, respectively. PS-3, designated as Fsp4 and Genetic Type Collection number T366H, is located on the terminus of MLG-F and linked to Sat 152 by 13.1 cM. PS-4, designated as Fsp5 and Genetic Type Collection number T367H, is located between SSR markers Satt324 and Satt138 on MLG-G and linked by 19.6 cM and 7.5 cM, respectively. SSR markers adjacent to Fsp3, Fsp4, and Fsp5 were distorted from a 1:2:1 ratio and fit a 1:1 ratio. The segregation distortions of SSR markers adjacent to Fsp3, Fsp4, and Fsp5 are in support of male, but not female transmission of the Fsp3, Fsp4, and Fsp5 gametes.This is a joint contribution of the Iowa Agriculture and Home Economics Experiment Station, Ames, Iowa, Project No. 3769 and from the USDA, Agricultural Research Service, Corn Insects and Crop Genetics Research Unit, and supported by Hatch Act and State of Iowa. The mention of a trademark or proprietary product does not constitute a guarantee or warranty of the product by Iowa State University or the USDA, and the use of the name by Iowa State University or the USDA implies no approval of the product to the exclusion of others that may also be suitable.Communicated by J. Dvorak     

Biochemical and molecular characterization of the Pseudomonas lemoignei polyhydroxyalkanoate depolymerase system.

Pseudomonas lemoignei has five different polyhydroxyalkanoate (PHA) depolymerase genes (phaZ1 to phaZ5), which encode the extracellularly localized poly(3-hydroxybutyrate) (PHB) depolymerases C, B, and D, poly(3-hydroxyvalerate) (PHV) depolymerase, and PHB depolymerase A, respectively. Four of the five genes (phaZ1 to phaZ4) have been cloned, and one of them (phaZ1) was studied in detail earlier (D. Jendrossek, B. Müller, and H. G. Schlegel, Eur. J. Biochem. 218:701-710, 1993). The fifth PHA depolymerase gene (phaZ5) was identified by colony hybridization of recombinant Escherichia coli clones with a phaZ5-specific oligonucleotide. The nucleotide sequence of a 3,704-bp EcoRI fragment was determined and found to contain two large open reading frames (ORFs) which coded for a polypeptide with significant similarities to glycerol-3-phosphate dehydrogenases of various sources (313 amino acids; M(r), 32,193) and for the precursor of PHB depolymerase A (PhaZ5; 433 amino acids; M(r), 44,906). The PHV depolymerase gene (phaZ4) was subcloned, and the nucleotide sequence of a 3,109-bp BamHI fragment was determined. Two large ORFs (ORF3 and ORF4) that represent putative coding regions were identified. The deduced amino acid sequence of ORF3 (134 amino acids; M(r), 14,686) revealed significant similarities to the branched-chain amino acid aminotransferase (IlfE) of enterobacteria. ORF4 (1,712 bp) was identified as the precursor of a PHV depolymerase (567 amino acids; M(r), 59,947). Analysis of primary structures of the five PHA depolymerases of P. lemoignei and of the PHB depolymerases of Alcaligenes faecalis and Pseudomonas pickettii revealed homologies of 25 to 83% to each other and a domain structure: at their N termini, they have typical signal peptides of exoenzymes. The adjacent catalytic domains are characterized by several conserved amino acids that constitute putative catalytic triads which consist of the consensus sequence of serine-dependent hydrolases including the pentapeptide G-X-S-X-G, a conserved histidine and aspartate, and a conserved region resembling the oxyanion hole of lipases. C terminal of the catalytic domain an approximately 40-amino-acid-long threonine-rich region (22 to 27 threonine residues) is present in PhaZ1, PhaZ2, PhaZ3, and PhaZ5. Instead of the threonine-rich region PhaZ4 and the PHB depolymerases of A. faecalis and P. pickettii contain an approximately 90-amino-acid-long sequence resembling the fibronectin type III module of eucaryotic extracellular matrix proteins. The function of the fibronectin type III module in PHA depolymerases remains obscure. Two types of C-terminal sequences apparently represent substrate-binding sites; the PHB type is present in the PHB depolymerases of A. faecalis and P. pickettii and in PhaZ2, PhaZ3, and PhaZ5 and the PHV type is present in the PHV-hydrolyzing depolymerases (PhaZ4 and PhaZ1). phaZ1 was transferred to A. eutrophus H16 and JMP222. All transconjugants of both strains were able to grow with extracellular PHB as a carbon source and produced translucent halos on PHB-containing solid media. PhaZ1, PhaZ2, PhaZ4, and PhaZ5 were purified from P. lemoignei and from recombinant E. coli; the processing sites of the precursors in E. coli were the same as in P. lemoignei, and similar substrate specificities were determined for the wild-type and the recombinant proteins. All PHA depolymerases hydrolyzed PHB at high specific activities. PhaZ1 and PhaZ4 additionally cleaved PHV, and PhaZ4 hydrolyzed poly(4-hydroxybutyrate). None of the depolymerases was able to hydrolyze polyactide or PHA consisting of monomers with more than five carbon atoms. While the wild-type depolymerase proteins were glycosylated and found to contain glucose and N-acetylglucosamine, none of the recombinant proteins was glycosylated. PHB hydrolysis was dependent on divalent cations such as Ca2+ and was inhibited by the presence of EDTA.     

Cloning, nucleotide sequence, and expression in Escherichia coli of the gene for poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis.

The extracellular poly(3-hydroxybutyrate) depolymerase gene from Alcaligenes faecalis T1 was cloned into Escherichia coli DH1 by using the plasmid pUC8. An A. faecalis T1 genomic library was prepared in E. coli from a partial Sau3AI digest and screened with antibody against the depolymerase. Of the 29 antibody-positive clones, 1 (pDP14), containing about 4 kilobase pairs of A. faecalis T1 DNA, caused expression of a high level of depolymerase activity in E. coli. The enzyme purified from E. coli was not significantly different from the depolymerase of A. faecalis in molecular weight, immunological properties, peptide map, specific activity, or substrate specificity. Most of the expressed enzyme was found to be localized in the periplasmic space of E. coli, although about 10% of the total activity was found in the culture medium. Results of a deletion experiment with pDP14 showed that a large SalI fragment of about 2 kilobase pairs was responsible for expression of the enzyme in E. coli. The nucleotide sequence of the large SalI fragment has been determined. Comparison of the deduced amino terminus with that obtained from sequence analysis of the purified protein indicated that poly(3-hydroxybutyrate) depolymerase exists as a 488-amino-acid precursor with a signal peptide of 27 amino acids.     

Synthesis of an Fmoc-Asn-heptasaccharide building block and its application to chemoenzymatic glycopeptide synthesis

The Fmoc-protected heptasaccharide asparagine building block β-GlcNAc-(1→2)-α-Man-(1→3)-[β-GlcNAc-(1→2)-α-Man-(1→6)]β-Man-(1→4)-β-GlcNAc-(1→4)-β-GlcNAc-(Fmoc)Asn was obtained by chemical synthesis. Two flexible strategies were developed with optimized conditions for the simultaneous debenzylation of the sugar and the amino acid part. The heptasaccharide asparagine building block is a partial structure of many glycoproteins and can be used for glycopeptide synthesis in solution and on the solid phase. In this work the heptasaccharide asparagine was elongated in solution to an Fmoc-glycopentapeptide methylester. After chemical cleavage of the Fmoc group the methylester was removed enzymatically by chymotrypsin. The use of β-(1→4)-galactosyltransferase and α-(2→6)-sialyltransferase in the presence of alkaline phosphatase allowed the efficient transfer of four sugar units to the acceptor resulting in an undecasaccharide glycopentapeptide.