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Query: UNIPROT:P06889 (Mol)
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Results from previous studies have suggested that an intramolecular disulphide bond in the exoprotein pullulanase is needed for its recognition and transport across the outer membrane. This interpretation of the data is shown here to be incorrect: pullulanase devoid of all potential disulphide bonds is secreted with apparently the same efficiency as the wild-type protein. Furthermore, the periplasmic disulphide bond, oxidoreductase DsbA, previously shown to catalyse the formation of a disulphide bond in pullulanase and to decrease its transit time in the periplasm, is shown here to be required for the rapid secretion of pullulanase devoid of disulphide bonds. Several possible explanations for the role of DsbA in pullulanase secretion are discussed.
Mol Microbiol 1998 Feb
PMID:The requirement for DsbA in pullulanase secretion is independent of disulphide bond formation in the enzyme. 948 77

Escherichia coli K-12 strains grown at 37 degrees C or 42 degrees C, but not at 30 degrees C, process the precursors of the Neisseria gonorrhoeae type IV pilin PilE and the Klebsiella oxytoca type IV pseudopilin PulG in a manner reminiscent of the prepilin peptidase-dependent processing of these proteins that occurs in these bacteria. Processing of prePulG in Escherichia coli requires a glycine at position -1, as does processing by the cognate prepilin peptidase (PulO), and is unaffected by mutations that inactivate several non-specific proteases. These data suggested that E. coli K-12 has a functional prepilin peptidase, despite the fact that it does not itself appear to express either type IV pilin or pseudopilin genes under the conditions that allow prePilE and prePulG processing. The E. coli K-12 genome contains two genes encoding proteins with significant sequence similarity to prepilin peptidases: gspO at minute 74.5 and pppA (f310c) at minute 67 on the genetic map. We have previously obtained evidence that gspO encodes an active enzyme but is not transcribed. pppA was cloned and shown to code for a functional prepilin peptidase capable of processing typical prepilin peptidase substrates. Inactivation of pppA eliminated the endogenous, thermoinducible prepilin peptidase activity. PppA was able to replace PulO prepilin peptidase in a pullulanase secretion system reconstituted in E. coli when expressed from high-copy-number plasmids but not when present in a single chromosomal copy. The analysis of pppA-lacZ fusions indicated that pppA expression was very low and regulated by the growth temperature at the level of translation, in agreement with the observed temperature dependence of PppA activity. Polymerase chain reaction and Southern hybridization analyses revealed the presence of the pppA gene in 12 out of 15 E. coli isolates.
Mol Microbiol 1998 Feb
PMID:A second prepilin peptidase gene in Escherichia coli K-12. 951 2

A decade ago, Pugsley and colleagues reported the existence of a large region of Klebsiella DNA, distinct from the Klebsiella gene encoding pullulanase, which was necessary for secretion of this enzyme to the cell surface in Escherichia coli (d'Enfert et al., 1987a,b). The pul genes it contained proved to be the tip of an iceberg. The sequences reported before 1992 (d'Enfert et al., 1987a,b; d'Enfert & Pugsley, 1989; Pugsley & Reyss, 1990; Reyss & Pugsley, 1990) included only one gene (pulD) that matched any sequence in the data base; a 220 amino acid residue segment of PulD was 32% identical with a portion of the filamentous phage-encoded protein, pIV. But by the time the sequence of the 18.8 kb DNA fragment that contained the pul genes had been completed (Possot et al., 1992), reports of sets of homologous genes in several species of Gram-negative plant and animal pathogens had appeared. For the most part, these gene clusters were cloned by their ability to complement mutants that produced, but failed to secrete, proteins normally found in the extracellular milieu; when tested, the mutants showed reduced pathogenicity or were totally avirulent. The secreted proteins included hydrolytic enzymes such as cellulase and pectinase from plant pathogens, and proteases and toxins from animal pathogens. The multi-gene family necessary for secretion of these enzymes is now known as the type II system or the main terminal branch (MTB) of the general secretion pathway (GSP). As summarized by Pugsley et al. (1997), the current tally includes type II systems from Klebsiella oxytoca (pul), Erwinia chrysanthemi and carotovora (out), Xanthomonas campestris (xps), Pseudomonas aeruginosa (xcp), Aeromonas hydrophila (exe), and Vibrio cholerae (eps). A second type II system (sps) necessary for deposition of the S-layer on the cell surface in A. hydrophila is more similar to the X. campestris than A. hydrophila genes (Thomas & Trust, 1995). The biggest surprise has been the discovery of a complete set of type II secretion genes in E. coli K12. The E. coli genes are not expressed under normal growth conditions, and a search is underway to find inducing conditions and secretion substrates (Francetic & Pugsley, 1996). Impressive progress has already been made in defining components of the pathway. What remains to be understood in mechanistic detail is how this protein secretion system functions.
J Mol Biol 1998 Jun 12
PMID:Macromolecular assembly and secretion across the bacterial cell envelope: type II protein secretion systems. 964 73

The notion of debranching enzyme activity as a participant in starch synthesis is gaining acceptance. Inconsistent reports from mutant analyses implicate either isoamylase or pullulanase as a determinant in amylopectin formation and whether wild-type plants utilize one or the other, or both, of these debranching enzymes in starch synthesis is unclear. Recent results on the sul mutant in maize suggest that both forms of debranching enzymes might be involved in amylopectin formation. We wished to find out if isoamylase takes part in starch synthesis by comparing isoamylase gene activity under three conditions: (1) during starch accumulation in developing sink tissues; (2) during starch degradation in germinating seeds; (3) in ectopic expression after applying sucrose, a starch precursor. We isolated the gene for barley isoamylase, isol, and analysed its expression and regulation in germinating seeds, developing endosperm and vegetative tissues, and compared the isoamylase gene expression in sink tissues from three different species. Our results indicate that isoamylase gene activity is involved in starch synthesis in wild-type plants and is modulated by sucrose.
Plant Mol Biol 1999 Jun
PMID:Analyses of isoamylase gene activity in wild-type barley indicate its involvement in starch synthesis. 1043 27

The RNA lariat debranching enzyme of mouse (mDBR1) was cloned by screening a NIH/3T3 cDNA library. The sequence of full-length mDBR1 cDNA contained a single 515 amino acid open reading frame of 58 kDa protein. Comparison of the amino acid sequence of mDBR1 to other DBR proteins showed 40%, 44%, 43%, 42%, and 80% identity to Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, Drosophila melanogaster, and human debranching enzymes, respectively. The mDBR1 cDNA was shown to be functional in an interspecies specific complementation experiment, and an in vitro debranching enzyme assay. Mouse DBR1 could complement the intron accumulation phenotype of a S. cerevisiae dbrl null mutant strain. However, the level of complementation depended on the copy number of the mDBR1 cDNA. The integration of the mDBR1 cDNA in the chromosome of S. pombe also complemented both intron accumulation and slow growth phenotypes of the S. pombe dbr1 knock-out mutant strain.
Mol Cells 2001 Apr 30
PMID:Cloning, expression, and complementation test of the RNA lariat debranching enzyme cDNA from mouse. 1135 1

Starch degrading enzymes, viz., beta-amylase, glucoamylase, and pullulanase, were purified using magnetite-alginate beads. In each case, the enzyme activity was eluted by using 1.0 M maltose. beta-Amylase (sweet potato), glucoamylase (Aspergillus niger), and pullulanase (Bacillus acidopullulyticus) from their crude preparations were purified 37-, 31-, and 49-fold with 86, 87, and 95% activity recovery, respectively. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis showed single band in each case.
Mol Biotechnol 2002 Mar
PMID:Magnetite-alginate beads for purification of some starch degrading enzymes. 1193 53

This review describes and discusses the implications of recent discoveries about how starch polymers are synthesized and organized to form a starch granule. Three issues are highlighted. 1. The role and importance of ADPglucose pyrophosphorylase in the generation of ADPglucose as the substrate for polymer synthesis. 2. The contributions of isoforms of starch-branching enzyme, starch synthase, and debranching enzyme to the synthesis and ordered packing of amylopectin molecules. 3. The requirements for and regulation of the synthesis of amylose.
Annu Rev Plant Physiol Plant Mol Biol 1997 Jun
PMID:THE SYNTHESIS OF THE STARCH GRANULE. 1501 57

The pseudopilin PulG is one of several essential components of the type II pullulanase secretion machinery (the Pul secreton) of the Gram-negative bacterium Klebsiella oxytoca. The sequence of the N-terminal 25 amino acids of the PulG precursor is hydrophobic and very similar to the corresponding region of type IV pilins. The structure of a truncated PulG (lacking the homologous region), as determined by X-ray crystallography, was found to include part of the long N-terminal alpha-helix and the four internal anti-parallel beta-strands that characterize type IV pilins, but PulG lacks the highly variable loop region with a disulphide bond that is found in the latter. When overproduced, PulG forms flexible pili whose structural features, as visualized by electron microscopy, are similar to those of bacterial type IV pili. The average helical repeat comprises 17 PulG subunits and four helical turns. Electron microscopy and molecular modelling show that PulG probably assembles into left-handed helical pili with the long N-terminal alpha-helix tightly packed in the centre of the pilus. As in the type IV pilins, the hydrophobic N-terminal part of the PulG alpha-helix is necessary for its assembly. Subtle sequence variations within this highly conserved segment seem to determine whether or not a type IV pilin can be assembled into pili by the Pul secreton.
Mol Microbiol 2004 Nov
PMID:Structure and assembly of the pseudopilin PulG. 1549 57

The crystal structures of Klebsiella pneumoniae pullulanase and its complex with glucose (G1), maltose (G2), isomaltose (isoG2), maltotriose (G3), or maltotetraose (G4), have been refined at around 1.7-1.9A resolution by using a synchrotron radiation source at SPring-8. The refined models contained 920-1052 amino acid residues, 942-1212 water molecules, four or five calcium ions, and the bound sugar moieties. The enzyme is composed of five domains (N1, N2, N3, A, and C). The N1 domain was clearly visible only in the structure of the complex with G3 or G4. The N1 and N2 domains are characteristic of pullulanase, while the N3, A, and C domains have weak similarity with those of Pseudomonas isoamylase. The N1 domain was found to be a new type of carbohydrate-binding domain with one calcium site (CBM41). One G1 bound at subsite -2, while two G2 bound at -1 approximately -2 and +2 approximately +1, two G3, -1 approximately -3 and +2 approximately 0', and two G4, -1 approximately -4 and +2 approximately -1'. The two bound G3 and G4 molecules in the active cleft are almost parallel and interact with each other. The subsites -1 approximately -4 and +1 approximately +2, including catalytic residues Glu706 and Asp677, are conserved between pullulanase and alpha-amylase, indicating that pullulanase strongly recognizes branched point and branched sugar residues, while subsites 0' and -1', which recognize the non-reducing end of main-chain alpha-1,4 glucan, are specific to pullulanase and isoamylase. The comparison suggested that the conformational difference around the active cleft, together with the domain organization, determines the different substrate specificities between pullulanase and isoamylase.
J Mol Biol 2006 Jun 09
PMID:Crystal structure of pullulanase: evidence for parallel binding of oligosaccharides in the active site. 1665 Aug 54

Starch recognition by carbohydrate-binding modules (CBMs) is important for the activity of starch-degrading enzymes. The N-terminal family 41 CBM, TmCBM41 (from pullulanase PulA secreted by Thermotoga maritima) was shown to have alpha-glucan binding activity with specificity for alpha-1,4-glucans but was able to tolerate the alpha-1,6-linkages found roughly every three or four glucose units in pullulan. Using X-ray crystallography, the structures were solved for TmCBM41 in an uncomplexed form and in complex with maltotetraose and 6(3)-alpha-D-glucosyl-maltotriose (GM3). Ligand binding was facilitated by stacking interactions between the alpha-faces of the glucose residues and two tryptophan side-chains in the two main subsites of the carbohydrate-binding site. Overall, this mode of starch binding is quite well conserved by other starch-binding modules. The structure in complex with GM3 revealed a third binding subsite with the flexibility to accommodate an alpha-1,4- or an alpha-1,6-linked glucose.
J Mol Biol 2007 Jan 19
PMID:The structural basis of alpha-glucan recognition by a family 41 carbohydrate-binding module from Thermotoga maritima. 1709 14


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