Purification and characterization of NADP-linked acetoacetyl-CoA reductase from Zoogloea ramigera I-16-M

An NADP-linked acetoacetyl-CoA reductase was purified to electrophoretic homogeneity from Zoogloea ramigera I-16-M, a poly(3-hydroxybutyrate)-accumulating bacterium. The purified enzyme showed specific activity of 412 mumol acetoacetyl-CoA reduced per min per mg protein, which constituted an 880-fold purification compared to the crude extract, with a 32% yield. Electrophoretic analysis of the purified enzyme which had been cross-linked with dimethylsuberimidate showed that the native enzyme (Mr 92,000) is a tetramer of four identical subunits (Mr 25,500). Among the various D-(-)- and L-(+)-3-hydroxyacyl-CoAs tested, the purified enzyme oxidized only D-(-)-3-hydroxybutyryl-CoA and to a lesser extent D-(-)-3-hydroxyvaleryl-CoA in the presence of NADP+. The antiserum prepared against the purified enzyme completely inhibited poly(3-hydroxybutyrate) synthesis from acetyl-CoA by a crude extract of Z. ramigera I-16-M cells. These findings indicate that this enzyme plays an indispensable role as the supplier of D-(-)-3-hydroxybutyryl-CoA in poly(3-hydroxybutyrate) synthesis in this bacterium.     

Analysis of β-ketothiolase and acetoacetyl-CoA reductase genes of a methylotrophic bacterium, Paracoccus denitrifleans, and their expression in Escherichia coli

Abstract The β-ketothiolase gene ( phaA ) and acetoacetyl-CoA reductase gene ( phaB ) were isolated from Paracoccus denitrificans . Nucleotide sequence analysis showed that they encoded proteins of 391 amino acids with a molecular mass of 40744 Da and of 242 amino acids with a molecular mass of 25614 Da, respectively. The predicted gene products exhibited high amino acid identities with those from other bacteria: 64.4–74.0% for the phaA gene product and 47.6–80.6% for the phaB gene product, respectively. Both genes were co-transcribed in a recombinant Escherichia coli . In addition, promoter activity was detected upstream of the phaA gene. Hence, the two genes are organized as an operon, phaA - phaB , in P. denitrificans . NADH was preferred to NADPH as a cofactor of acetoacetyl-CoA reductase.     

The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hydroxybutyryl-CoA for poly(D-3-hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an alpha 4 homotetramer of four 25-kDa subunits. It has a Km of 2 microM and a kcat/Km of 1.8 X 10(8) M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 microM. K is 10(-10) M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3',5'-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase.     

Nucleotide sequence of the gene encoding the active-site serine β-lactamase from Streptomyces cacaoi

Abstract The gene encoding the extracellular active-site serine β-lactamase of Streptomyces cacaoi previously cloned into Streptomyces lividans , has the information for the synthesis of a 303 amino-acid precursor. The β-lactamase as excreted by the host S. lividans ML1, has a ragged amino-terminus, indicating either the presence of a leader peptidase of poor specificity or the action of an amino-peptidase. The deduced primary structure has been confirmed by amino acid sequencing of a 10-residue stretch at the amino terminus of the mature protein and an 8-residue stretch containing the active-site serine. The S. cacaoi β-lactamase is highly homologous with the class A β-lactamases of Streptomyces albus G and Staphylococcus aureus of known three-dimensional structure. Amino acid alignments show that the S. cacaoi β-lactamase essentially differs from these two latter enzymes by short insertions and deletions that do not affect the spatial disposition of the secondary structures.     

Nucleotide sequence of the gene encoding the active-site serine β-lactamase from Actinomadura R39

The gene encoding the extracellular, active-site serine beta-lactamase of Actinomadura R39, previously cloned into Streptomyces lividans, has the information for the synthesis of a 304 amino acid protein, the amino terminal region of which has the characteristic features of a signal peptide. The Actinomadura R39 beta-lactamase is another member of the class A beta-lactamases. In particular, it shows high homology with the beta-lactamase of Bacillus licheniformis.     

Formaldehyde kills spores of Bacillus subtilis by DNA damage and small, acid-soluble spore proteins of the ααααααα/βββββββ-type protect spores against this DNA damage

Killing of wild-type spores of Bacillus subtilis with formaldehyde also caused significant mutagenesis; spores (termed α⁻β⁻) lacking the two major α/β-type small, acid-soluble spore proteins (SASP) were more sensitive to both formaldehyde killing and mutagenesis. A recA mutation sensitized both wild-type and α⁻β⁻ spores to formaldehyde treatment, which caused significant expression of a recA - lacZ fusion when the treated spores germinated. Formaldehyde also caused protein–DNA cross-linking in both wild-type and α⁻β⁻ spores. These results indicate that: (i) formaldehyde kills B. subtilis spores at least in part by DNA damage and (b) α/β-type SASP protect against spore killing by formaldehyde, presumably by protecting spore DNA.     

Purification and partial amino acid sequence of plantaricin 1.25ααα and 1.25βββ, two bacteriocins produced by Lactobacillus plantarum TMW1.25

Two bacteriocins produced by Lactobacillus plantarum TMW1.25 have been purified by a four-step purification procedure, including ammonium sulphate precipitation and cation-exchange chromatography followed by hydrophobic-interaction chromatography on octyl sepharose. The final purification was performed by repeated reversed-phase chromatography steps which yielded two bacteriocin fractions designated plantaricin 1.25 alpha and plantaricin 1.25 beta. The molecular masses of the peptides in these fractions were 5979 and 5203 Da, respectively. Combination of the fractions did not have any synergistic effects on bacteriocin activity, indicating that they each contain a one-peptide bacteriocin. The major peptide in the alpha fraction was blocked at its N-terminus, and a partial sequence (25 residues) could only be obtained after cleavage with CNBr. This sequence did not show clear homologies with known bacteriocins. The beta peptide has been sequenced almost completely and consists, presumably, of 53 residues. This peptide displayed strong homology to the known N-terminal part of brevicin 27 produced by Lactobacillus brevis SB27. The results showed that the beta peptide contains as many as six consecutive lysine residues at the N-terminus.     

The sequence of porcine β-casein cDNA

Porcine cDNAs clones encoding beta-casein were isolated and sequenced. The porcine beta-casein cDNA is 1100bp in length, excluding the poly(A) tail, and encodes a preprotein of 232 amino acids.     

Cloning and nucleotide sequence of the gene encoding the γ-d-glutamyl-l-diamino acid endopeptidase II of Bacillus sphaericus

The gene encoding the Bacillus sphaericus gamma-D-glutamyl-L-diamino acid endopeptidase II, a cytoplasmic enzyme involved in cell sporulation [1], contains the information for a 271-amino acid protein devoid of a signal peptide. The endopeptidase lacks sequence relatedness with other proteins of known primary structure except that its C-terminal region has significant similarity with the C-terminal region of the 54-kDa P54 protein of Enterococcus faecium, of unknown function [2].     

Identification of TaqI polymorphism in the mitochondrial acetoacetyl-CoA thiolase gene and familial analysis of 3-ketothiolase deficiency

We analyzed the mitochondrial acetoacetyl-CoA thiolase gene (T2) by Southern blotting. Fifteen unrelated healthy individuals and members of five families with 3-ketothiolase deficiency (3KTD) were analyzed. We found a TaqI polymorphism, the heterozygosity of which was calculated to be 0.5 among healthy Japanese individuals. This restriction fragment length polymorphism (RFLP) proved to be useful for detecting 3KTD patients and its obligatory carriers, at the DNA level and in two out of five 3KTD families. This polymorphism was found to be generated by the presence/ absence of a TaqI site in intron 9 of the T2 gene. With in vitro amplification of the genomic region around the TaqI site, this RFLP can be detected within 2 days.     

Immunohistochemical Localization of G Protein β1, β2, β3, β4, β5, and γ3 Subunits in the Adult Rat Brain

Abstract: The regional distributions of the G protein β subunits (Gβ1–β5) and of the Gγ3 subunit were examined by immunohistochemical methods in the adult rat brain. In general, the Gβ and Gγ3 subunits were widely distributed throughout the brain, with most regions containing several Gβ subunits within their neuronal networks. The olfactory bulb, neocortex, hippocampus, striatum, thalamus, cerebellum, and brainstem exhibited light to intense Gβ immunostaining. Negative immunostaining was observed in cortical layer I for Gβ1 and layer IV for Gβ4. The hippocampal dentate granular and CA1–CA3 pyramidal cells displayed little or no positive immunostaining for Gβ2 or Gβ4. No anti-Gβ4 immunostaining was observed in the pars compacta of the substantia nigra or in the cerebellar granule cell layer and Purkinje cells. Immunoreactivity for Gβ1 was absent from the cerebellar molecular layer, and Gβ2 was not detected in the Purkinje cells. No positive Gγ3 immunoreactivity was observed in the lateral habenula, lateral septal nucleus, or Purkinje cells. Double-fluorescence immunostaining with anti-Gγ3 antibody and individual anti-Gβ1–β5 antibodies displayed regional selectivity with Gβ1 (cortical layers V–VI) and Gβ2 (cortical layer I). In conclusion, despite the widespread overlapping distributions of Gβ1–β5 with Gγ3, specific dimeric associations in situ were observed within discrete brain regions.     

Cloning and nucleotide sequence of the structural gene encoding for human tryptophanyl-tRNA synthetase.

A structural gene encoding bovine (b) tryptophanyl-tRNA synthetase (WRS) has recently been cloned and sequenced [Garret et al., Biochemistry 30 (1991) 7809-7817]. Using part of this sequence as a hybridisation probe we have cloned and sequenced a structural gene encoding human polypeptide highly homologous with two mammalian proteins, bWRS [Garret et al., Biochemistry 30 (1991) 7809-7817; EMBL accession No. X52113] and rabbit peptide chain release factor [Lee et al., Proc. Natl. Acad. Sci. USA 87 (1990) 3508-3512]. Identification of the sequence encoding a human WRS is based on (i) the presence of 'HIGH' and 'KMSKS' structural motifs typical for class-I aminoacyl-tRNA synthetases [Eriani et al., Nature 347 (1990) 203-206]; (ii) coincidence of the number of SH groups per subunit estimated experimentally [Muench et al., Science 187 (1975) 1089-1091] and deduced from the cDNA sequence (six in both cases); (iii) close resemblance of two WRS polypeptides sequenced earlier [Muench et al., Science 187 (1975) 1089-1091] and the predicted structure in two different regions.     

Cloning and nucleotide sequence of the chlD locus

The nucleotide sequence of a Sau3A1 restriction nuclease fragment that complemented an Escherichia coli chlD::Mu cts mutant strain was determined. DNA and deduced amino acid sequence analysis revealed two open reading frames (ORFs) that potentially codes for proteins with amino acid sequence homology with binding protein-dependent transport systems. One of the ORFs showed a sequence that encoded a protein with properties that were characteristic of a hydrophobic inner membrane protein. The other ORF, which was responsible for complementing a chlD mutant, encoded a protein with conserved sequences in nucleotide-binding proteins and hydrophilic inner membrane proteins in active transport systems. A proposal that the chlD locus is the molybdate transport operon is discussed in terms of the chlD phenotype.     

Cloning and nucleotide sequence of the bglA gene from Erwinia herbicola and expression of β-glucosidase activity in Escherichia coli

Abstract Genomic DNA fragments encoding β-glucosidase activity from the wild-type strain WD4 of Erwinia herbicola were cloned into Escherichia coli . Two clones containing a common fragment encoded a polypeptide of 58000 Da. Cloned β-glucosidase, expressed in E. coli , showed activity against natural β-glucoside sugars except for cellobiose. An open reading frame of 1442 bp termed bglA was identified by nucleotide sequencing and it coded for a protein of 480 amino acids ( M _r 53896) which showed significant homology with β-glucosidases from glycosyl hydrolase family 1.     

Partial nucleotide sequence of a novel bovine major histocompatibility complex class II β-chain gene, BoLA-DIB

A bovine genomic clone that hybridized to HLA-DQ beta cDNA was isolated and fragments containing the beta 1, beta 2 and transmembrane (TM) exons subcloned. The nucleotide sequences of the exons and flanking intron regions were determined. Comparisons of these exon nucleotide sequences and derived amino acid sequences to human class II beta-chain sequences showed that this gene is only 77% identical to HLA-DQ beta and about 75% identical to bovine DQ beta-like genes. The exon sequences were more divergent from other class II beta-chain genes. However, structural features such as conserved cysteines and regions of amino acids strongly suggest this to be a class II beta-chain gene. When exon-containing fragments were used as hybridization probes on Southern blots of bovine genomic DNA digested with Eco RI or Pvu II, each exon hybridized to a single band. Based on these results we have referred to this gene as a novel bovine class II beta-chain gene, BoLA-DIB.