Unusual Starch Degradation Pathway via Cyclodextrins in the Hyperthermophilic Sulfate-Reducing Archaeon Archaeoglobus fulgidus Strain 7324

Abstract

The hyperthermophilic archaeon Archaeoglobus fulgidus strain 7324 has been shown to grow on starch and sulfate and thus represents the first sulfate reducer able to degrade polymeric sugars. The enzymes involved in starch degradation to glucose 6-phosphate were studied. In extracts of starch-grown cells the activities of the classical starch degradation enzymes, ␣-amylase and amylopullulanase, could not be detected. Instead, evidence is presented here that A. fulgidus utilizes an unusual pathway of starch degradation involving cyclodextrins as intermediates. The pathway comprises the combined action of an extracellular cyclodextrin glucanotransferase (CGTase) converting starch to cyclodextrins and the intracellular conversion of cyclodextrins to glucose 6-phosphate via cyclodextrinase (CDase), maltodextrin phosphorylase (Mal-P), and phosphoglucomutase (PGM). These enzymes, which are all induced after growth on starch, were characterized. CGTase catalyzed the conversion of starch to mainly ␤-cyclodextrin. The gene encoding CGTase was cloned and sequenced and showed highest similarity to a glucanotransferase from Thermococcus litoralis. After transport of the cyclodextrins into the cell by a transport system to be defined, these molecules are linearized via a CDase, catalyzing exclusively the ring opening of the cyclodextrins to the respective maltooligodextrins. These are degraded by a Mal-P to glucose 1-phosphate. Finally, PGM catalyzes the conversion of glucose 1-phosphate to glucose 6-phosphate, which is further degraded to pyruvate via the modified Embden-Meyerhof pathway.

Sequence fingerprints of enzyme specificities from the glycoside hydrolase family GH57

“…Sequences were collected based on basic protein BLAST (http://blast.ncbi.nlm.nih.gov/Blast.cgi) (Altschul et al 1990) searches using the complete sequences of 14 experimentally characterized GH57 enzymes: a-amylase from Methanocaldococcus jannaschii (Bult et al 1996;Kim et al 2001;Li and Peeples 2004), amylopullulanases from Pyrococcus furiosus (Dong et al 1997;Kang et al 2005), Thermococcus hydrothermalis (Erra-Pujada et al 1999), Thermococcus litoralis ) and Thermococcus siculi (Jiao et al 2011), branching enzymes from Thermococcus kodakaraensis (Murakami et al 2006;Santos et al 2011), Thermotoga maritima Dickmanns et al 2006) and Thermus thermophilus (Palomo et al 2011), 4-a-glucanotransferases from Archaeoglobus fulgidus (Labes and Schonheit 2007), Dictyoglomus thermophilum (Fukusumi et al 1988;Nakajima et al 2004), T. kodakaraensis (Tachibana et al 1997(Tachibana et al , 2000, T. litoralis (Jeon et al 1997) and P. furiosus (Laderman et al 1993a, b), and a-galactosidase from P. furiosus (van Lieshout et al 2003).…”

Two potentially novel amylolytic enzyme specificities in the prokaryotic glycoside hydrolase α-amylase family GH57

“…a-Helices and b-strands (predicted by the Phyre-2 server; Kelley & Sternberg, 2009) are coloured in red and green, respectively. Abbreviations: AMY_Uncba, non-specified amylase from uncultured bacterium ; MGA_Pycfu, maltogenic amylase from P. furiosus (Comfort et al, 2008); AAMY_Mccja, a-amylase from M. jannaschii (Kim et al, 2001); PAMY_Bctth, putative aamylase-like protein from B. thetaiotaomicron (Janeč ek & Blesá k, 2011); APU_Pycfu, APU_Thchy, APU_Thcli and APU_Thcsi, amylopullulanases from P. furiosus (Dong et al, 1997), Thermococcus hydrothermalis (Erra-Pujada et al, 1999), Thermococcus litoralis and Thermococcus siculi (Jiao et al, 2011), respectively; APCD_Sttma, amylopullulanase-cyclomaltodextrinase from S. marinus (Li et al, 2013); BE_Thcko, BE_Thtma and BE_Theth, branching enzymes from Thermococcus kodakaraensis (Murakami et al, 2006), Thermotoga maritima and Thermus thermophilus (Palomo et al, 2011), respectively;4AGT_Arcfu, 4AGT_Dicth, 4AGT_Pycfu, 4AGT_Thcko, 4AGT_Thcli, 4-a-glucanotransferases from Archaeoglobus fulgidus (Labes & Schö nheit, 2007), D. thermophilum (Fukusumi et al, 1988), P. furiosus (Laderman et al, 1993a), Thermococcus kodakaraensis (Tachibana et al, 1997) and Thermococcus litoralis (Jeon et al, 1997), respectively; AGAL_Pycfu, a-galactosidase from P. furiosus (van Lieshout et al, 2003). CSRs are emphasized by rectangles and catalytic residues (CSR-3 glutamate, catalytic nucleophile; CSR-4 aspartate, proton donor) are indicated by asterisks.…”

Identification and Characterization of an Archaeal Kojibiose Catabolic Pathway in the Hyperthermophilic Pyrococcus sp. Strain ST04

“…strain B1001 and Archaeoglobus fulgidus, genes for two cyclodextrin-utilizing enzymes, cyclodextrin glucanotransferase (CGTase) and cyclodextrinase (CDase), together with an ABC transporter cluster were found in the genome. In these strains, cyclodextrin is formed by CGTase using starch, then assimilated into the cell through an ABC transporter system, and completely hydrolyzed into glucose and maltose by CDase (2,36). Until now, the utilization of other disaccharides, such as kojibiose, in the archaea has not been reported.…”

“…They are commonly composed of extracellular hydrolases, transporter complexes, and regulatory systems (1,2). Among these, maltose and maltodextrin uptake systems have been widely investigated (3,4).…”