Transcriptional and biochemical analysis of starch metabolism in the hyperthermophilic archaeon Pyrococcus furiosus.
Pyrococcus furiosus utilizes starch and its degradation products, such as maltose, as primary carbon sources, but the pathways by which these alpha-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-alpha-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 alpha-glucan substrates by P. furiosus.
Lee
Han-Seung
HS
Department of Biochemistry and Molecular Biology, University of Georgia, Athens, GA 30602-7229, USA.
Shockley
Keith R
KR
Schut
Gerrit J
GJ
Conners
Shannon B
SB
Montero
Clemente I
CI
Johnson
Matthew R
MR
Chou
Chung-Jung
CJ
Bridger
Stephanie L
SL
Wigner
Nathan
N
Brehm
Scott D
SD
Jenney
Francis E
FE
Comfort
Donald A
DA
Kelly
Robert M
RM
Adams
Michael W W
MW
D019843:Archaeal Proteins / Q000235:genetics; D001426:Bacterial Proteins / Q000235:genetics; D019641:DNA, Archaeal / Q000737:chemistry / Q000235:genetics; D020869:Gene Expression Profiling; D019848:Gene Expression Regulation, Archaeal; D005947:Glucose / Q000378:metabolism; D005958:Glucosephosphates / Q000378:metabolism; D005964:Glucosyltransferases / Q000235:genetics; D006004:Glycogen Debranching Enzyme System / Q000235:genetics; D006026:Glycoside Hydrolases / Q000235:genetics; D009004:Monosaccharide Transport Proteins / Q000235:genetics; D020411:Oligonucleotide Array Sequence Analysis; D020027:Pyrococcus furiosus / Q000235:genetics / Q000378:metabolism*; D017422:Sequence Analysis, DNA; D013213:Starch / Q000378:metabolism*; D013379:Substrate Specificity
D019843:Archaeal Proteins; D001426:Bacterial Proteins; D019641:DNA, Archaeal; D005958:Glucosephosphates; D006004:Glycogen Debranching Enzyme System; D009004:Monosaccharide Transport Proteins; D013213:Starch; C031590:glucose-1-phosphate; D005964:Glucosyltransferases; C033938:maltodextrin phosphorylase; C020569:4 alpha-glucanotransferase; D006026:Glycoside Hydrolases; C082507:amylopullulanase; D005947:Glucose