Transcriptional and Biochemical Analysis of Starch Metabolism in the Hyperthermophilic Archaeon Pyrococcus furiosus

Abstract

Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these ␣-glucans are processed have yet to be defined. For example, its genome contains genes proposed to encode five amylolytic enzymes (including a cyclodextrin glucanotransferase [CGTase] and amylopullulanase), as well as two transporters for maltose and maltodextrins (Mal-I and Mal-II), and a range of intracellular enzymes have been purified that reportedly metabolize maltodextrins and maltose. However, precisely which of these enzymes are involved in starch processing is not clear. In this study, starch metabolism in P. furiosus was examined by biochemical analyses in conjunction with global transcriptional response data for cells grown on a variety of glucans. In addition, DNA sequencing led to the correction of two key errors in the genome sequence, and these change the predicted properties of amylopullulanase (now designated PF1935*) and CGTase (PF0478*). Based on all of these data, a pathway is proposed that is specific for starch utilization that involves one transporter (Mal-II [PF1933 to PF1939]) and only three enzymes, amylopullulanase (PF1935*), 4-␣-glucanotransferase (PF0272), and maltodextrin phosphorylase (PF1535). Their expression is upregulated on starch, and together they generate glucose and glucose-1-phosphate, which then feed into the novel glycolytic pathway of this organism. In addition, the results indicate that several hypothetical proteins encoded by three gene clusters are also involved in the transport and processing of ␣-glucan substrates by P. furiosus.Pyrococcus furiosus is an obligately anaerobic, heterotrophic, hyperthermophilic archaeon that was isolated from a shallow hydrothermal vent near Vulcano Island, Italy (12). The organism grows optimally near 100°C and is able to utilize a range of sugars as a primary carbon source (4,11,28,29). These include cellobiose (28, 43), laminarin (43), chitin (16), maltose (29), barley glucan (3), and starch (29). However, the means by which these compounds are metabolized by P. furiosus are not well understood. In attempting to define the pathway by which starch, and one of its degradation products, maltose, serve as carbon sources, there are two complicating factors. First, the genome sequence of P. furiosus (36) contains an array of genes that encode putative glycosyl hydrolases and glycosyl transferases. With respect to starch, they include five enzymes that potentially hydrolyze ␣-glucans. Two of them (PF0477 and PF1935) have putative signal peptides and are presumably extracellular, while the other three (PF0272, PF0478, and PF1939) lack a signal peptide and are presumably intracellular. They are annotated as amylases (PF0272 and PF0477), amylopullulanases (PF1934 and PF1935), and cyclodextrin glucanotransferases (PF0478). The question is, are all of these enzymes involved in degrading starch? The pathway for utilizing maltose and maltodextrins, primary products of starch breakdown, is well defined in ...

Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance

“…4H, we also found that Apu Tk plays a much greater role than expected in the cleavage of ␣-1,4-glycosidic linkages, suggesting that the experimentally verified ␣-amylase TK1884 protein (59) may not be the major amylose-degrading enzyme in T. kodakaraensis. This agrees well with the results of a transcriptome analysis of P. furiosus grown on starch, which revealed that PF1935*, the homolog of TK1774*, is the protein most up-regulated in the presence of starch (39).…”

Genome Sequence of Thermofilum pendens Reveals an Exceptional Loss of Biosynthetic Pathways without Genome Reduction

“…The enzymes involved in sucrose metabolism could not be identified from the genome sequence. No beta-fructofuranosidase (invertase) or sucrose phosphorylase could be identified, and there is no homolog of PF0132, which encodes the invertase purified from P. furiosus (27).…”

A Novel Branching Enzyme of the GH-57 Family in the Hyperthermophilic Archaeon Thermococcus kodakaraensis KOD1

“…According to the literature, four enzyme activities have been detected in the family (18,46). The activities are ␣-amylase (22, 27), 4-␣-glucanotransferase (19,26,29,35), amylopullulanase (5,7,8), and ␣-galactosidase (43). The TK1436 protein was incubated at 70°C with melibiose, pullulan, maltoheptaose, or amylose as the substrate, and the reaction mixtures were analyzed by TLC (Fig.…”