Purification, characterization, and gene cloning of a novel maltosyltransferase from an Arthrobacter globiformis strain that produces an alternating alpha-1,4- and alpha-1,6-cyclic tetrasaccharide from starch.
A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM; cyclo-{-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->6)-alpha-D-Glcp(1-->4)-alpha-D-Glcp(1-->}] from starch, was purified to homogeneity from the culture supernatant of Arthrobacter globiformis M6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50 degrees C and was stable from pH 5.0 to 9.0 and up to 30 degrees C. The addition of 1 mM Ca2+ enhanced the thermal stability of the enzyme up to 45 degrees C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of > or =3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose --> 6(4)-O-alpha-maltosyl-maltotetraose + maltose and (ii) 6(4)-O-alpha-maltosyl-maltotetraose --> CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular alpha-1,6-maltosyl transfer reactions. The gene for 6MT, designated cmmA, was isolated from a genomic library of A. globiformis M6. The cmmA gene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to alpha-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the alpha-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.
Mukai
Kazuhisa
K
Amase Institute, Hayashibara Biochemical Laboratories, Inc., 7-7 Amase minami-machi, Okayama 700-0834, Japan. k-mukai@hayashibara.co.jp
Watanabe
Hikaru
H
Kubota
Michio
M
Chaen
Hiroto
H
Fukuda
Shigeharu
S
Kurimoto
Masashi
M
D000595:Amino Acid Sequence; D001173:Arthrobacter / Q000201:enzymology* / Q000235:genetics*; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D004269:DNA, Bacterial / Q000235:genetics; D004795:Enzyme Stability; D005798:Genes, Bacterial; D005964:Glucosyltransferases / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008954:Models, Biological; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009844:Oligosaccharides / Q000096:biosynthesis* / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D012988:Soil Microbiology; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
D004269:DNA, Bacterial; D009844:Oligosaccharides; D013213:Starch; D005964:Glucosyltransferases; C436710:maltosyltransferase