Dataset Information

Metabolism Dealing with Thermal Degradation of NAD⁺ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

ABSTRACT: 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.

SUBMITTER: Hachisuka SI

PROVIDER: S-EPMC5585711 | biostudies-literature | 2017 Oct

REPOSITORIES: biostudies-literature

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Metabolism Dealing with Thermal Degradation of NAD+ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Hachisuka Shin-Ichi SI Sato Takaaki T Atomi Haruyuki H

Journal of bacteriology 20170905 19

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+ r ...[more]

PMID: 28652302

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Project description:Background: Prokaryotes have relatively small genomes, densely-packed and apparently dominated by protein-encoding sequences. However, data now generated by high throughput RNA sequencing (RNA-seq) reveal surprisingly more-complex transcriptomes with many previously unrecognized and unanticipated non-coding small and antisense transcripts. To date, such studies have investigated primarily Bacteria. Here, we report the transcripts present in Thermococcus kodakarensis, a model hyperthermophilic Archaeon, synthesized under different growth and metabolic conditions. Results: cDNA libraries, generated from RNA preparations isolated from cells growing in media with sulfur or pyruvate, with sulfur to stationary phase, and growing with pyruvate but with sulfur added 20 min before RNA isolation, have been deep-sequenced. The results identify >2,700 sites of transcription initiation, establish a genome-wide map of transcripts, and consensus sequences for transcription initiation and post-transcription regulatory elements in T. kodakarensis. Primary transcription start sites (TSS) are identified upstream of 1,254 annotated genes, including ~78 % of those predicted by promoter locations, and an additional 644 primary TSS and their promoters have been identified within genes. Most of the mRNAs have a 5'-untranslated region (5'-UTR) between 10 and 50 nt long (median length = 16 nt), ~20 % have 5'-UTRs from 50 to 300 nt long, ~14 % are leaderless with 5'-UTRs ≤8 nt, and ~50% contain a consensus ribosome binding sequence. The results also identify TSS for 1,018 antisense transcripts, most with sequences complementary to either the 5'- or 3'-region of a sense mRNA. The data confirm the presence of transcripts from all three CRISPR loci, the RNase P and 7S RNAs, all tRNAs and rRNAs and 69 snoRNAs predicted to be encoded in the T. kodakarensis genome. Two transcripts, putatively identified as riboswitches, were present in RNA preparations isolated from growing but not from stationary phase cells. The procedure used is designed to identify TSS but, assuming that the number of cDNA reads correlates with transcript abundance, the data obtained also provide a semi-quantitative overview of global operon expression. They document substantial differences in gene expression under different physiological conditions and are consistent with previous observations of substrate-dependent specific gene expression. Many previously unrecognized and 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. Conclusion: We have identified >2,700 TSS that include almost all of the primary sites of transcription initiation upstream of annotated genes, and also 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 results reveal the presence of many non-coding RNAs and predict extensive RNA-based regulation in T. kodakarensis.

Dataset Information

Metabolism Dealing with Thermal Degradation of NAD⁺ in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

Publications

Metabolism Dealing with Thermal Degradation of NAD<sup>+</sup> in the Hyperthermophilic Archaeon Thermococcus kodakarensis.

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