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ABSTRACT Ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) plays a central role in carbon dioxide fixation on our planet. Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis ( Tk-Rubisco) shows approximately twenty times the activity of spinach Rubisco at high temperature, but only one-eighth the activity at ambient temperature. We have tried to improve the activity of Tk-Rubisco at ambient temperature, and have successfully constructed several mutants which showed higher activities than the wild-type enzyme both in vitro and in vivo. Here, we designed new Tk-Rubisco mutants based on its three-dimensional structure and a sequence comparison of thermophilic and mesophilic plant Rubiscos. Four mutations were introduced to generate new mutants based on this strategy, and one of the four mutants, T289D, showed significantly improved activity compared to that of the wild-type enzyme. The crystal structure of the Tk-Rubisco T289D mutant suggested that the increase in activity was due to mechanisms distinct from those involved in the improvement in activity of Tk-Rubisco SP8, a mutant protein previously reported to show the highest activity at ambient temperature. Combining the mutations of T289D and SP8 successfully generated a mutant protein (SP8-T289D) with the highest activity to date both in vitro and in vivo. The improvement was particularly pronounced for the in vivo activity of SP8-T289D when introduced into the mesophilic, photosynthetic bacterium Rhodopseudomonas palustris, which resulted in a strain with nearly two-fold higher specific growth rates compared to that of a strain harboring the wild-type enzyme at ambient temperature. Proteins 2016; 84:1339-1346. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

A [NiFe] hydrogenase maturation protease HybD from Thermococcus kodakarensis KOD1 (TkHybD) is involved in the cleavage of the C-terminal residues of [NiFe] hydrogenase large subunits by Ni recognition. Here, we report the crystal structure of TkHybD at 1.82 Å resolution to better understand this process. TkHybD exhibits an a/β/a sandwich fold with conserved residues responsible for the Ni recognition. Comparisons of TkHybD with homologous proteins also reveal that they share a common overall architecture, suggesting that they have similar catalytic functions. Our results including metal binding site prediction provide insight into the substrate recognition and catalysis mechanism of TkHybD. [ABSTRACT FROM AUTHOR]

The TK2203 protein from the hyperthermophilic archaeon Thermococcus kodakarensis KOD1 (262 residues, 29 kDa) is a putative extradiol dioxygenase catalyzing the cleavage of C-C bonds in catechol derivatives. It contains three metal-binding residues, but has no significant sequence similarity to proteins for which structures have been determined. Here, the first crystal structure of the TK2203 protein was determined at 1.41 Å resolution to investigate its functional role. Structure analysis reveals that this protein shares the same fold and catalytic residues as other extradiol dioxygenases, strongly suggesting the same enzymatic activity. Furthermore, the important region contributing to substrate selectivity is discussed. [ABSTRACT FROM AUTHOR]

Coenzyme A (CoA) plays pivotal roles in a variety of metabolic pathways in all organisms. The biosynthetic pathway of CoA is strictly regulated by feedback inhibition. In the hyperthermophilic archaeon Thermococcus kodakarensis, ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate, is a target of feedback inhibition by CoA. The crystal structure of KPR from T. kodakarensis (Tk-KPR) complexed with CoA and 2-oxopantoate has previously been reported. The structure provided an explanation for the competitive inhibition mechanism. Here, further biochemical analyses of Tk-KPR and the crystal structure of Tk-KPR in complex with NADP+ are reported. A mutational analysis implies that the residues in the binding pocket cooperatively contribute to the recognition of CoA. The structure reveals the same dimer architecture as the Tk-KPR-CoA-2-oxopantoate complex. Moreover, the positions of the residues involved in the dimer interaction are not changed by the binding of CoA and 2-oxopantoate, suggesting individual conformational changes of Tk-KPR monomers. [ABSTRACT FROM AUTHOR]

ABSTRACT Coenzyme A (CoA) plays essential roles in a variety of metabolic pathways in all three domains of life. The biosynthesis pathway of CoA is strictly regulated by feedback inhibition. In bacteria and eukaryotes, pantothenate kinase is the target of feedback inhibition by CoA. Recent biochemical studies have identified ketopantoate reductase (KPR), which catalyzes the NAD(P)H-dependent reduction of 2-oxopantoate to pantoate, as a target of the feedback inhibition by CoA in archaea. However, the mechanism for recognition of CoA by KPR is still unknown. Here we report the crystal structure of KPR from Thermococcus kodakarensis in complex with CoA and 2-oxopantoate. CoA occupies the binding site of NAD(P)H, explaining the competitive inhibition by CoA. Our structure reveals a disulfide bond between CoA and Cys84 that indicates an irreversible inhibition upon binding of CoA. The structure also suggests the cooperative binding of CoA and 2-oxopantoate that triggers a conformational closure and seems to facilitate the disulfide bond formation. Our findings provide novel insights into the mechanism that regulates biosynthesis of CoA in archaea. Proteins 2016; 84:374-382. © 2016 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

Proper protein folding is an essential process for all organisms. Prefoldin (PFD) is a molecular chaperone that assists protein folding by delivering non-native proteins to group II chaperonin. A heterohexamer of eukaryotic PFD has been shown to specifically recognize and deliver non-native actin and tubulin to chaperonin-containing TCP-1 (CCT), but the mechanism of specific recognition is still unclear. To determine its crystal structure, recombinant human PFD was reconstituted, purified and crystallized. X-ray diffraction data were collected to 4.7 Å resolution. The crystals belonged to space group P21212, with unit-cell parameters a = 123.2, b = 152.4, c = 105.9 Å. [ABSTRACT FROM AUTHOR]

UDP- N-acetylglucosamine pyrophosphorylase (UAP) is an essential enzyme in the synthesis of UDP- N-acetylglucosamine. UAP from Candida albicans was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals of the substrate and product complexes both diffract X-rays to beyond 2.3 Å resolution using synchrotron radiation. The crystals of the substrate complex belong to the triclinic space group P1, with unit-cell parameters a = 47.77, b = 62.89, c = 90.60 Å, α = 90.01, β = 97.72, γ = 92.88°, whereas those of the product complex belong to the orthorhombic space group P212121, with unit-cell parameters a = 61.95, b = 90.87, c = 94.88 Å. [ABSTRACT FROM AUTHOR]

N-acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthesis of UDP- N-acetylglucosamine (UDP-GlcNAc) in eukaryotes and belongs to the α-d-phosphohexomutase superfamily. AGM1 from Candida albicans (CaAGM1) was purified and crystallized by the sitting-drop vapour-diffusion method. The crystals obtained belong to the primitive monoclinic space group P21, with unit-cell parameters a = 60.2, b = 130.2, c = 78.0 Å, β = 106.7°. The crystals diffract X-rays to beyond 1.8 Å resolution using synchrotron radiation. [ABSTRACT FROM AUTHOR]