Entire nucleotide sequence of the pullulanase gene of Klebsiella aerogenes W70

Abstract

We determined the entire nucleotide sequence of the Klebsiella aerogenes W70 pullulanase gene (pulA) contained on a 4.2-kilobase-pair fragment of plasmid pPB174. The amino acid composition deduced from an open reading frame of 3,288 base pairs agreed closely with that determined for the intracellular pullalanase. The precursor enzyme consisted of 1,096 amino acid residues and contained a hydrophobic N-terminal signal peptide and the consensus sequence for the bacterial prelipoprotein signal peptide cleavage site.Pullulanase (EC 3.2.1.41, pullulan 6-glucanohydrolase), a starch-debranching enzyme, hydrolyzes (1-6)-a-glucosidic linkages in pullulan and starch to form maltotriose. This enzyme is produced by the gram-negative bacterium Kleb-0 1.0 genes W70 (19). We sequenced the entire pullulanase gene (pulA) and showed that the precursor enzyme contained N-terminal signal peptide. The precursor and mature proteins deduced from the DNA sequence showed a high degree

Nucleotide sequence of the maltotetraohydrolase gene from Pseudomonas saccharophila

“…When the entire primary sequence of the G4-forming amylase was compared with those of other amylolytic enzymes (various a-amylases [4][5][6][7][8], two cyclomaltodextrin glucanotransferases [12,13], maltohexaohydrolase [14], isoamylase [15], and pullulanase [16]), the sequence similarities were extremely weak (data not shown); the percent identity was 26.3°70 for barley aamylase [5], but was less than 9070 for all other amylolytic enzymes examined. However, three regions (domains I, II, and III), which constitute both the catalytic and substrate-binding sites of the amylolytic enzymes [24], are highly conserved in the sequence of the G4-forming amylase (residues 112-117, 189-197, and 288-295, respectively, see fig.3).…”

“…A 3.1-kbp DNA fragment encoding the G4-forming amylase [17] contains two open reading frames, ORF-1 and ORF-2, the former of which actually encodes the G4-forming amylase gene (figs 1 and G6-forming amylase [14]; human saliva gland u-amylase [4]; barley c~-amylase [5]; Aspergillus oryzae a-amylase [6]; Bacillus amyloliquefaciens t~-amylase [7]; Bacillus subtilis t~-amylase [8]; Bacillus stearothermophilus cyclomaltodextrin glucanotransferase (CGTase) [12]; Klebsiella pneumoniae CGTase [13]; Pseudomonus amyloderamosa isoamylase [15]; Klebsiella aerogenes pullulanase [16].…”

“…The amino acid sequences of various amylolytic enzymes such as u-amylase [4][5][6][7][8],/~-amylase [9], glucoamylase [10], cyclomaltodextrin glucanotransferase [11][12][13], maltohexaohydrolase [14], isoamylase [15], and pullulanase [16], have been determined by direct protein analysis or deduced from the DNA sequences. It is of great interest to study the molecular basis for the difference between the specificity of the G4-forming amylase and those of the other amylolytic enzymes.…”

A Single Limit Dextrinase Gene Is Expressed Both in the Developing Endosperm and in Germinated Grains of Barley1

“…A second role that has been suggested for limit dextrinase is in starch synthesis in the (Fig. 2), rice (Nakamura et al, 1996), spinach (A. Renz, R. Schmid, J. Kossmann, and E. Beck, unpublished data), and K. aerogenes (Katsuragi et al, 1987), using the PileUp and PrettyBox programs (Devereux et al, 1984). Shaded and hatched boxes indicate identical and homologous residues, respectively.…”

“…X83969; A. Renz, R. Schmid, J. Kossmann, and E. Beck, unpublished data), a pullulanase from the bacterium Klebsiella aerogenes (39% identity; Katsuragi et al, 1987), the sugary1 gene of maize (24% identity; James et al, 1995), and an isoamylase from Pseudomonas amyloderamosa (25% identity; Amemura et al, 1988). The alignment of these sequences, shown in Figure 8, is restricted to the catalytic site region, but blocks of approximately 2 to 10 identical amino acids are also distributed along the rest of the polypeptide chain.…”