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Structure of a D-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima.

ABSTRACT: The crystal structure of a D-tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2 A. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of D-tagatose 3-epimerase from Pseudomonas cichorii and D-psicose 3-epimerase from Agrobacterium tumefaciens; however, TM0416p exhibited a unique solvent-accessible substrate-binding pocket that reflected the absence of an alpha-helix that covers the active-site cleft in the two aforementioned ketohexose 3-epimerases. In addition, the residues responsible for creating a hydrophobic environment around the substrate in TM0416p differ entirely from those in the other two enzymes. Collectively, these findings suggest that the substrate specificity of TM0416p is likely to differ substantially from those of other D-tagatose 3-epimerase family enzymes.

SUBMITTER: Sakuraba H

PROVIDER: S-EPMC2650453 | biostudies-literature | 2009 Mar

REPOSITORIES: biostudies-literature

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Structure of a D-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima.

Sakuraba Haruhiko H Yoneda Kazunari K Satomura Takenori T Kawakami Ryushi R Ohshima Toshihisa T

Acta crystallographica. Section F, Structural biology and crystallization communications 20090214 Pt 3

The crystal structure of a D-tagatose 3-epimerase-related protein (TM0416p) encoded by the hypothetical open reading frame TM0416 in the genome of the hyperthermophilic bacterium Thermotoga maritima was determined at a resolution of 2.2 A. The asymmetric unit contained two homologous subunits and a dimer was generated by twofold symmetry. The main-chain coordinates of the enzyme monomer proved to be similar to those of D-tagatose 3-epimerase from Pseudomonas cichorii and D-psicose 3-epimerase fr ...[more]

PMID: 19255464

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Project description:UnlabelledRegulation of coenzyme A (CoA) biosynthesis in bacteria and eukaryotes occurs through feedback inhibition targeting type I and type II pantothenate kinase (PanK), respectively. In contrast, the activity of type III PanK is not affected by CoA. As the hyperthermophilic bacterium Thermotoga maritima harbors only a single type III PanK (Tm-PanK), here we examined the mechanisms that regulate CoA biosynthesis in this organism. We first examined the enzyme responsible for the ketopantoate reductase (KPR) reaction, which is the target of feedback inhibition in archaea. A classical KPR homolog was not present on the T. maritima genome, but we found a homolog (TM0550) of the ketol-acid reductoisomerase (KARI) from Corynebacterium glutamicum, which exhibits KPR activity. The purified TM0550 protein displayed both KPR and KARI activities and was designated Tm-KPR/KARI. When T. maritima cell extract was subjected to anion-exchange chromatography, the fractions containing high levels of KPR activity also displayed positive signals in a Western blot analysis using polyclonal anti-TM0550 protein antisera, strongly suggesting that Tm-KPR/KARI was the major source of KPR activity in the organism. The KPR activity of Tm-KPR/KARI was not inhibited in the presence of CoA. We thus examined the properties of Tm-PanK and the pantothenate synthetase (Tm-PS) of this organism. Tm-PS was not affected by CoA. Surprisingly however, Tm-PanK was inhibited by CoA, with almost complete inhibition in the presence of 400 μM CoA. Our results suggest that CoA biosynthesis in T. maritima is regulated by feedback inhibition targeting PanK, although Tm-PanK is a type III enzyme.ImportanceBacteria and eukaryotes regulate the biosynthesis of coenzyme A (CoA) by feedback inhibition targeting type I or type II pantothenate kinase (PanK). The hyperthermophilic bacterium Thermotoga maritima harbors a single type III PanK (Tm-PanK), previously considered to be unaffected by CoA. By examining the properties of three enzymes involved in CoA biosynthesis in this organism, we found that Tm-PanK, although a type III enzyme, is inhibited by CoA. The results provide a feasible explanation of how CoA biosynthesis is regulated in T. maritima, which may also apply for other bacteria that harbor only type III PanK enzymes.

Project description:Thermotoga maritima is an anaerobic hyperthermophilic bacterium that efficiently produces H2 by fermenting carbohydrates. High concentration of H2 inhibits the growth of T. maritima, and S0 could eliminate the inhibition and stimulate the growth through its reduction. The mechanism of T. maritima sulfur reduction, however, has not been fully understood. Herein, based on its similarity with archaeal NAD(P)H-dependent sulfur reductases (NSR), the ORF THEMA_RS02810 was identified and expressed in Escherichia coli, and the recombinant protein was characterized. The purified flavoprotein possessed NAD(P)H-dependent S0 reductase activity (1.3 U/mg for NADH and 0.8 U/mg for NADPH), polysulfide reductase activity (0.32 U/mg for NADH and 0.35 U/mg for NADPH), and thiosulfate reductase activity (2.3 U/mg for NADH and 2.5 U/mg for NADPH), which increased 3~4-folds by coenzyme A stimulation. Quantitative RT-PCR analysis showed that nsr was upregulated together with the mbx, yeeE, and rnf genes when the strain grew in S0- or thiosulfate-containing medium. The mechanism for sulfur reduction in T. maritima was discussed, which may affect the redox balance and energy metabolism of T. maritima. Genome search revealed that NSR homolog is widely distributed in thermophilic bacteria and archaea, implying its important role in the sulfur cycle of geothermal environments. IMPORTANCE The reduction of S0 and thiosulfate is essential in the sulfur cycle of geothermal environments, in which thermophiles play an important role. Despite previous research on some sulfur reductases of thermophilic archaea, the mechanism of sulfur reduction in thermophilic bacteria is still not clearly understood. Herein, we confirmed the presence of a cytoplasmic NAD(P)H-dependent polysulfide reductase (NSR) from the hyperthermophile T. maritima, with S0, polysulfide, and thiosulfate reduction activities, in contrast to other sulfur reductases. When grown in S0- or thiosulfate-containing medium, its expression was upregulated. And the putative membrane-bound MBX and Rnf may also play a role in the metabolism, which might influence the redox balance and energy metabolism of T. maritima. This is distinct from the mechanism of sulfur reduction in mesophiles such as Wolinella succinogenes. NSR homologs are widely distributed among heterotrophic thermophiles, suggesting that they may be vital in the sulfur cycle in geothermal environments.

Dataset Information

Structure of a D-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima.

Publications

Structure of a D-tagatose 3-epimerase-related protein from the hyperthermophilic bacterium Thermotoga maritima.

Similar Datasets

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