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. 1994 Sep;60(9):3096–3104. doi: 10.1128/aem.60.9.3096-3104.1994

Properties and active center of the thermostable branching enzyme from Bacillus stearothermophilus.

H Takata 1, T Takaha 1, T Kuriki 1, S Okada 1, M Takagi 1, T Imanaka 1
PMCID: PMC201776  PMID: 7944355

Abstract

Although the branching enzyme (EC 2.4.1.18) is a member of the alpha-amylase family, the characteristics are not understood. The thermostable branching enzyme gene from Bacillus stearothermophilus TRBE14 was cloned and expressed in Escherichia coli. The branching enzyme was purified to homogeneity, and various enzymatic properties were analyzed by our improved assay method. About 80% of activity was retained when the enzyme was heated at 60 degrees C for 30 min, and the optimum temperature for activity was around 50 degrees C. The enzyme was stable in the range of pH 7.5 to 9.5, and the optimum pH was 7.5. The nucleotide sequence of the gene was determined, and the active center of the enzyme was analyzed by means of site-directed mutagenesis. The catalytic residues were tentatively identified as two Asp residues and a Glu residue by comparison of the amino acid sequences of various branching enzymes from different sources and enzymes of the alpha-amylase family. When the Asp residues and Glu were replaced by Asn and Gln, respectively, the branching enzyme activities disappeared. The results suggested that these three residues are the catalytic residues and that the catalytic mechanism of the branching enzyme is basically identical to that of alpha-amylase. On the basis of these results, four conserved regions including catalytic residues and most of the substrate-binding residues of various branching enzymes are proposed.

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Selected References

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  1. Baba T., Kimura K., Mizuno K., Etoh H., Ishida Y., Shida O., Arai Y. Sequence conservation of the catalytic regions of amylolytic enzymes in maize branching enzyme-I. Biochem Biophys Res Commun. 1991 Nov 27;181(1):87–94. doi: 10.1016/s0006-291x(05)81385-3. [DOI] [PubMed] [Google Scholar]
  2. Bachmann B. J. Pedigrees of some mutant strains of Escherichia coli K-12. Bacteriol Rev. 1972 Dec;36(4):525–557. doi: 10.1128/br.36.4.525-557.1972. [DOI] [PMC free article] [PubMed] [Google Scholar]
  3. Baecker P. A., Greenberg E., Preiss J. Biosynthesis of bacterial glycogen. Primary structure of Escherichia coli 1,4-alpha-D-glucan:1,4-alpha-D-glucan 6-alpha-D-(1, 4-alpha-D-glucano)-transferase as deduced from the nucleotide sequence of the glg B gene. J Biol Chem. 1986 Jul 5;261(19):8738–8743. [PubMed] [Google Scholar]
  4. Boyer C., Preiss J. Biosynthesis of bacterial glycogen. Purification and properties of the Escherichia coli b alpha-1,4,-glucan: alpha-1,4-glucan 6-glycosyltansferase. Biochemistry. 1977 Aug 9;16(16):3693–3699. doi: 10.1021/bi00635a029. [DOI] [PubMed] [Google Scholar]
  5. Fisher D. K., Boyer C. D., Hannah L. C. Starch branching enzyme II from maize endosperm. Plant Physiol. 1993 Jul;102(3):1045–1046. doi: 10.1104/pp.102.3.1045. [DOI] [PMC free article] [PubMed] [Google Scholar]
  6. Govons S., Vinopal R., Ingraham J., Preiss J. Isolation of mutants of Escherichia coli B altered in their ability to synthesize glycogen. J Bacteriol. 1969 Feb;97(2):970–972. doi: 10.1128/jb.97.2.970-972.1969. [DOI] [PMC free article] [PubMed] [Google Scholar]
  7. Imanaka T., Fujii M., Aramori I., Aiba S. Transformation of Bacillus stearothermophilus with plasmid DNA and characterization of shuttle vector plasmids between Bacillus stearothermophilus and Bacillus subtilis. J Bacteriol. 1982 Mar;149(3):824–830. doi: 10.1128/jb.149.3.824-830.1982. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. Imanaka T., Kuriki T. Pattern of action of Bacillus stearothermophilus neopullulanase on pullulan. J Bacteriol. 1989 Jan;171(1):369–374. doi: 10.1128/jb.171.1.369-374.1989. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Jespersen H. M., MacGregor E. A., Sierks M. R., Svensson B. Comparison of the domain-level organization of starch hydrolases and related enzymes. Biochem J. 1991 Nov 15;280(Pt 1):51–55. doi: 10.1042/bj2800051. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Kiel J. A., Boels J. M., Beldman G., Venema G. Molecular cloning and nucleotide sequence of the glycogen branching enzyme gene (glgB) from Bacillus stearothermophilus and expression in Escherichia coli and Bacillus subtilis. Mol Gen Genet. 1991 Nov;230(1-2):136–144. doi: 10.1007/BF00290661. [DOI] [PubMed] [Google Scholar]
  11. Kiel J. A., Boels J. M., Beldman G., Venema G. The glgB gene from the thermophile Bacillus caldolyticus encodes a thermolabile branching enzyme. DNA Seq. 1992;3(4):221–232. doi: 10.3109/10425179209034021. [DOI] [PubMed] [Google Scholar]
  12. Kiel J. A., Elgersma H. S., Beldman G., Vossen J. P., Venema G. Cloning and expression of the branching enzyme gene (glgB) from the cyanobacterium Synechococcus sp. PCC7942 in Escherichia coli. Gene. 1989 May 15;78(1):9–17. doi: 10.1016/0378-1119(89)90309-0. [DOI] [PubMed] [Google Scholar]
  13. Kossmann J., Visser R. G., Müller-Röber B., Willmitzer L., Sonnewald U. Cloning and expression analysis of a potato cDNA that encodes branching enzyme: evidence for co-expression of starch biosynthetic genes. Mol Gen Genet. 1991 Nov;230(1-2):39–44. doi: 10.1007/BF00290648. [DOI] [PubMed] [Google Scholar]
  14. Kozak M. Comparison of initiation of protein synthesis in procaryotes, eucaryotes, and organelles. Microbiol Rev. 1983 Mar;47(1):1–45. doi: 10.1128/mr.47.1.1-45.1983. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Kuriki T., Okada S., Imanaka T. New type of pullulanase from Bacillus stearothermophilus and molecular cloning and expression of the gene in Bacillus subtilis. J Bacteriol. 1988 Apr;170(4):1554–1559. doi: 10.1128/jb.170.4.1554-1559.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
  16. Kuriki T., Takata H., Okada S., Imanaka T. Analysis of the active center of Bacillus stearothermophilus neopullulanase. J Bacteriol. 1991 Oct;173(19):6147–6152. doi: 10.1128/jb.173.19.6147-6152.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  17. Matsuura Y., Kusunoki M., Harada W., Kakudo M. Structure and possible catalytic residues of Taka-amylase A. J Biochem. 1984 Mar;95(3):697–702. doi: 10.1093/oxfordjournals.jbchem.a134659. [DOI] [PubMed] [Google Scholar]
  18. Messing J. New M13 vectors for cloning. Methods Enzymol. 1983;101:20–78. doi: 10.1016/0076-6879(83)01005-8. [DOI] [PubMed] [Google Scholar]
  19. Mizuno K., Kawasaki T., Shimada H., Satoh H., Kobayashi E., Okumura S., Arai Y., Baba T. Alteration of the structural properties of starch components by the lack of an isoform of starch branching enzyme in rice seeds. J Biol Chem. 1993 Sep 5;268(25):19084–19091. [PubMed] [Google Scholar]
  20. Mizuno K., Kimura K., Arai Y., Kawasaki T., Shimada H., Baba T. Starch branching enzymes from immature rice seeds. J Biochem. 1992 Nov;112(5):643–651. doi: 10.1093/oxfordjournals.jbchem.a123953. [DOI] [PubMed] [Google Scholar]
  21. Nakamura Y., Yamanouchi H. Nucleotide sequence of a cDNA encoding starch-branching enzyme, or q-enzyme I, from rice endosperm. Plant Physiol. 1992 Jul;99(3):1265–1266. doi: 10.1104/pp.99.3.1265. [DOI] [PMC free article] [PubMed] [Google Scholar]
  22. Okita T. W., Rodriguez R. L., Preiss J. Biosynthesis of bacterial glycogen. Cloning of the glycogen biosynthetic enzyme structural genes of Escherichia coli. J Biol Chem. 1981 Jul 10;256(13):6944–6952. [PubMed] [Google Scholar]
  23. Poulsen P., Kreiberg J. D. Starch branching enzyme cDNA from Solanum tuberosum. Plant Physiol. 1993 Jul;102(3):1053–1054. doi: 10.1104/pp.102.3.1053. [DOI] [PMC free article] [PubMed] [Google Scholar]
  24. Romeo T., Kumar A., Preiss J. Analysis of the Escherichia coli glycogen gene cluster suggests that catabolic enzymes are encoded among the biosynthetic genes. Gene. 1988 Oct 30;70(2):363–376. doi: 10.1016/0378-1119(88)90208-9. [DOI] [PubMed] [Google Scholar]
  25. Rumbak E., Rawlings D. E., Lindsey G. G., Woods D. R. Characterization of the Butyrivibrio fibrisolvens glgB gene, which encodes a glycogen-branching enzyme with starch-clearing activity. J Bacteriol. 1991 Nov;173(21):6732–6741. doi: 10.1128/jb.173.21.6732-6741.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
  26. Takata H., Kuriki T., Okada S., Takesada Y., Iizuka M., Minamiura N., Imanaka T. Action of neopullulanase. Neopullulanase catalyzes both hydrolysis and transglycosylation at alpha-(1----4)- and alpha-(1----6)-glucosidic linkages. J Biol Chem. 1992 Sep 15;267(26):18447–18452. [PubMed] [Google Scholar]
  27. Thon V. J., Khalil M., Cannon J. F. Isolation of human glycogen branching enzyme cDNAs by screening complementation in yeast. J Biol Chem. 1993 Apr 5;268(10):7509–7513. [PubMed] [Google Scholar]
  28. Thon V. J., Vigneron-Lesens C., Marianne-Pepin T., Montreuil J., Decq A., Rachez C., Ball S. G., Cannon J. F. Coordinate regulation of glycogen metabolism in the yeast Saccharomyces cerevisiae. Induction of glycogen branching enzyme. J Biol Chem. 1992 Jul 25;267(21):15224–15228. [PubMed] [Google Scholar]

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