Maltose-forming α-amylase from the hyperthermophilic archaeon Pyrococcus sp. ST04
- 585 Downloads
The deduced amino acid sequence from a gene of the hyperthermophilic archaeon Pyrococcus sp. ST04 (Py04_0872) contained a conserved glycoside hydrolase family 57 (GH57) motif, but showed <13 % sequence identity with other known Pyrococcus GH57 enzymes, such as 4-α-glucanotransferase (EC 184.108.40.206), amylopullulanase (EC 220.127.116.11), and branching enzyme (EC 18.104.22.168). This gene was cloned and expressed in Escherichia coli, and the recombinant product (P yrococcus sp. ST04 maltose-forming α-amylase, PSMA) was a novel 70-kDa maltose-forming α-amylase. PSMA only recognized maltose (G2) units with α-1,4 and α-1,6 linkages in polysaccharides (e.g., starch, amylopectin, and glycogen) and hydrolyzed pullulan very poorly. G2 was the primary end product of hydrolysis. Branched cyclodextrin (CD) was only hydrolyzed along its branched maltooligosaccharides. 6-O-glucosyl-β-cyclodextrin (G1-β-CD) and β-cyclodextrin (β-CD) were resistant to PSMA suggesting that PSMA is an exo-type glucan hydrolase with α-1,4- and α-1,6-glucan hydrolytic activities. The half-saturation value (K m) for the α-1,4 linkage of maltotriose (G3) was 8.4 mM while that of the α-1,6 linkage of 6-O-maltosyl-β-cyclodextrin (G2-β-CD) was 0.3 mM. The k cat values were 381.0 min−1 for G3 and 1,545.0 min−1 for G2-β-CD. The enzyme was inhibited competitively by the reaction product G2, and the K i constant was 0.7 mM. PSMA bridges the gap between amylases that hydrolyze larger maltodextrins and α-glucosidase that feeds G2 into glycolysis by hydrolyzing smaller glucans into G2 units.
KeywordsPyrococcus sp. ST04 Hyperthermophile Maltose-forming α-amylase Maltose inhibition
This work was supported by a National Research Foundation of Korea (NRF) grant funded by the Korean government (MEST; no. 2012–0005289).
- Choi HC, Seo DH, Jung JH, Ha SJ, Kim MJ, Lee JH, Chang PS, Kim HY, Park CS (2011) Development of new assay for sucrose phosphorylase and its application to the characterization of Bifidobacterium longum SJ32 sucrose phosphorylase. Food Sci Biotechnol 20(2):513–518. doi: 10.1007/s10068-011-0071-0 CrossRefGoogle Scholar
- Comfort DA, Chou CJ, Conners SB, VanFossen AL, Kelly RM (2008) Functional-genomics-based identification and characterization of open reading frames encoding α-glucoside-processing enzymes in the hyperthermophilic archaeon Pyrococcus furiosus. Appl Environ Microbiol 74(4):1281–1283PubMedCentralPubMedCrossRefGoogle Scholar
- Jung JH, Lee JH, Holden JF, Seo DH, Shin H, Kim HY, Kim W, Ryu S, Park CS (2012) Complete genome sequence of the hyperthermophilic archaeon Pyrococcus sp. strain ST04, isolated from a deep-sea hydrothermal sulfide chimney on the Juan de Fuca Ridge. J Bacteriol 194(16):4434–4435PubMedCentralPubMedCrossRefGoogle Scholar
- Koga S, Yoshioka I, Sakuraba H, Takahashi M, Sakasegawa S, Shimizu S, Ohshima T (2000) Biochemical characterization, cloning, and sequencing of ADP-dependent (AMP-forming) glucokinase from two hyperthermophilic archaea, Pyrococcus furiosus and Thermococcus litoralis. J Biochem 128(6):1079–1085PubMedCrossRefGoogle Scholar
- Lee HS, Shockley KR, Schut GJ, Conners SB, Montero CI, Johnson MR, Chou CJ, Bridger SL, Wigner N, Brehm SD, Jenney FE Jr, Comfort DA, Kelly RM, Adams MW (2006) Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus. J Bacteriol 188(6):2115–2125PubMedCentralPubMedCrossRefGoogle Scholar
- Lee JH, Karamychev VN, Kozyavkin SA, Mills D, Pavlov AR, Pavlova NV, Polouchine NN, Richardson PM, Shakhova VV, Slesarev AI, Weimer B, O’Sullivan DJ (2008) Comparative genomic analysis of the gut bacterium Bifidobacterium longum reveals loci susceptible to deletion during pure culture growth. BMC Genomics 9:247PubMedCentralPubMedCrossRefGoogle Scholar
- Li X, Li D, Park KH (2013) An extremely thermostable amylopullulanase from Staphylothermus marinus displays both pullulan- and cyclodextrin-degrading activities. Appl Microbiol Biotechnol 97(12):5359–5369Google Scholar
- Palomo M, Pijning T, Booiman T, Dobruchowska JM, van der Vlist J, Kralj S, Planas A, Loos K, Kamerling JP, Dijkstra BW, van der Maarel MJ, Dijkhuizen L, Leemhuis H (2011) Thermus thermophilus glycoside hydrolase family 57 branching enzyme: crystal structure, mechanism of action, and products formed. J Biol Chem 286(5):3520–3530PubMedCrossRefGoogle Scholar
- Santos CR, Tonoli CC, Trindade DM, Betzel C, Takata H, Kuriki T, Kanai T, Imanaka T, Arni RK, Murakami MT (2011) Structural basis for branching-enzyme activity of glycoside hydrolase family 57: structure and stability studies of a novel branching enzyme from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1. Proteins 79(2):547–557PubMedCrossRefGoogle Scholar