Dataset Information

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

ABSTRACT: A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved in the degradation of CDs (cgtB), the gene encoding CGTase (cgtA), a gene homolog for a CD-binding protein (CBP) (cgtC), and a putative CBP-dependent ABC transporter involved in uptake of CDs (cgtDE). The CDase was expressed in Escherichia coli and purified. The optimum pH and temperature for CD hydrolysis were 5.5 and 95 degrees C, respectively. The molecular weight of the recombinant enzyme was estimated to be 79,000. The CDase hydrolyzed beta-CD most efficiently among other CDs. Maltose and pullulan were not utilized as substrates. Linear maltodextrins with a small glucose unit were very slowly hydrolyzed, and starch was hydrolyzed more slowly. Analysis by thin-layer chromatography revealed that glucose and maltose were produced as end products. The purified recombinant CBP bound to maltose as well as to alpha-CD. However, the CBP exhibited higher thermostability in the presence of alpha-CD. These results suggested that strain B1001 possesses a unique metabolic pathway that includes extracellular synthesis, transmembrane uptake, and intracellular degradation of CDs in starch utilization. Potential advantages of this starch metabolic pathway via CDs are discussed.

SUBMITTER: Hashimoto Y

PROVIDER: S-EPMC95380 | biostudies-literature | 2001 Sep

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

Hashimoto Y Y Yamamoto T T Fujiwara S S Takagi M M Imanaka T T

Journal of bacteriology 20010901 17

A unique extracellular and thermostable cyclomaltodextrin glucanotransferase (CGTase) from the hyperthermophilic archaeon Thermococcus sp. strain B1001 produces predominantly (>85%) alpha-cyclomaltodextrin (alpha-CD) from starch (Y. Tachibana, et al., Appl. Environ. Microbiol. 65:1991--1997, 1999). Nucleotide sequencing of the CGTase gene (cgtA) and its flanking region was performed, and a cluster of five genes was found, including a gene homolog encoding a cyclomaltodextrinase (CDase) involved ...[more]

PMID: 11489857

Similar Datasets

Project description:NAD+ is an important cofactor for enzymatic oxidation reactions in all living organisms, including (hyper)thermophiles. However, NAD+ is susceptible to thermal degradation at high temperatures. It can thus be expected that (hyper)thermophiles harbor mechanisms that maintain in vivo NAD+ concentrations and possibly remove and/or reuse undesirable degradation products of NAD+ Here we confirmed that at 85°C, thermal degradation of NAD+ results mostly in the generation of nicotinamide and ADP-ribose, the latter known to display toxicity by spontaneously linking to proteins. The hyperthermophilic archaeon Thermococcus kodakarensis possesses a putative ADP-ribose pyrophosphatase (ADPR-PPase) encoded by the TK2284 gene. ADPR-PPase hydrolyzes ADP-ribose to ribose 5-phosphate (R5P) and AMP. The purified recombinant TK2284 protein exhibited activity toward ADP-ribose as well as ADP-glucose. Kinetic analyses revealed a much higher catalytic efficiency toward ADP-ribose, suggesting that ADP-ribose was the physiological substrate. To gain insight into the physiological function of TK2284, a TK2284 gene disruption strain was constructed and examined. Incubation of NAD+ in the cell extract of the mutant strain at 85°C resulted in higher ADP-ribose accumulation and lower AMP production compared with those in experiments with the host strain cell extract. The mutant strain also exhibited lower cell yield and specific growth rates in a synthetic amino acid medium compared with those of the host strain. The results obtained here suggest that the ADPR-PPase in T. kodakarensis is responsible for the cleavage of ADP-ribose to R5P and AMP, providing a means to utilize the otherwise dead-end product of NAD+ breakdown.IMPORTANCE Hyperthermophilic microorganisms living under high temperature conditions should have mechanisms that deal with the degradation of thermolabile molecules. NAD+ is an important cofactor for enzymatic oxidation reactions and is susceptible to thermal degradation to ADP-ribose and nicotinamide. Here we show that an ADP-ribose pyrophosphatase homolog from the hyperthermophilic archaeon Thermococcus kodakarensis converts the detrimental ADP-ribose to ribose 5-phosphate and AMP, compounds that can be directed to central carbon metabolism. This physiological role for ADP-ribose pyrophosphatases might be universal in hyperthermophiles, as their homologs are widely distributed among both hyperthermophilic bacteria and archaea.

Project description:Prokaryotes have relatively small genomes, densely-packed with protein-encoding sequences. RNA sequencing has, however, revealed surprisingly complex transcriptomes and here we report the transcripts present in the model hyperthermophilic Archaeon, Thermococcus kodakarensis, under different physiological conditions.Sequencing cDNA libraries, generated from RNA isolated from cells under different growth and metabolic conditions has identified >2,700 sites of transcription initiation, established a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements. The primary transcription start sites (TSS) upstream of 1,254 annotated genes, plus 644 primary TSS and their promoters within genes, are identified. Most mRNAs have a 5'-untranslated region (5'-UTR) 10 to 50 nt long (median = 16 nt), but ~20% have 5'-UTRs from 50 to 300 nt long and ~14% are leaderless. Approximately 50% of mRNAs contain a consensus ribosome binding sequence. The results identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA, and confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 predicted snoRNAs. Two putative riboswitch RNAs were present in growing but not in stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the results also provide a semi-quantitative documentation of the differences in T. kodakarensis genome expression under different growth conditions and confirm previous observations of substrate-dependent specific gene expression. Many previously unanticipated small RNAs have been identified, some with relative low GC contents (? 50%) and sequences that do not fold readily into base-paired secondary structures, contrary to the classical expectations for non-coding RNAs in a hyperthermophile.The results identify >2,700 TSS, including almost all of the primary sites of transcription initiation upstream of annotated genes, plus many secondary sites, sites within genes and sites resulting in antisense transcripts. The T.?kodakarensis genome is small (~2.1 Mbp) and tightly packed with protein-encoding genes, but the transcriptomes established also contain many non-coding RNAs and predict extensive RNA-based regulation in this model Archaeon.

Dataset Information

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

Publications

Extracellular synthesis, specific recognition, and intracellular degradation of cyclomaltodextrins by the hyperthermophilic archaeon Thermococcus sp. strain B1001.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets