Eriko Matsui

Novel substrate specificity of a membrane-bound β-glycosidase from the hyperthermophilic archaeon Pyrococcus horikoshii

A β‐glycosidase gene homolog of Pyrococcus horikoshii (BGPh) was successfully expressed in Escherichia coli. The enzyme was localized in a membrane fraction and solubilized with 2.5% Triton X‐100 at 85°C for 15 min. The optimum pH was 6.0 and the optimum temperature was over 100°C, respectively. BGPh stability was dependent on the presence of Triton X‐100, the enzymes half‐life at 90°C (pH 6.0) was 15 h. BGPh has a novel substrate specificity with k cat/K m values high enough for hydrolysis of β‐D‐Glcp derivatives with long alkyl chain at the reducing end and low enough for the hydrolysis of β‐linked glucose dimer more hydrophilic than aryl‐ or alkyl‐β‐D‐Glcp.

Molecular Structure and Novel DNA Binding Sites Located in Loops of Flap Endonuclease-1 from Pyrococcus horikoshii

The crystal structure of flap endonuclease-1 fromPyrococcus horikoshii (phFEN-1) was determined to a resolution of 3.1 Å. The active cleft of the phFEN-1 molecule is formed with one large loop and four small loops. We examined the function of the conserved residues and positively charged clusters on these loops by kinetic analysis with 45 different mutants. Arg40 and Arg42 on small loop 1, a cluster Lys193–Lys195 on small loop 2, and two sites, Arg94 and Arg118-Lys119, on the large loop were identified as binding sites. Lys87 on the large loop may play significant roles in catalytic reaction. Furthermore, we successfully elucidated the function of the four DNA binding sites that form productive ES complexes specific for each endo- or exo-type hydrolysis, probably by bending the substrates. For the endo-activity, Arg94 and Lys193–Lys195 located at the top and bottom of the molecule were key determinants. For the exo-activity, all four sites were needed, but Arg118-Lys119 was dominant. The major binding sites for both the nick substrate and double-stranded DNA might be the same.

Novel structure of an N-terminal domain that is crucial for the dimeric assembly and DNA-binding of an archaeal DNA polymerase D large subunit from Pyrococcus horikoshii

DP2 binds to DP2 by circular dichroism (View interaction)

The novel function of a short region K253xRxxxD259 conserved in the exonuclease domain of hyperthermostable DNA polymerase I from Pyrococcus horikoshii.

Abstract. The DNA polymerase gene of the hyperthermophile Pyrococcus horikoshii was successfully overexpressed after removing an intein. The importance of an amino acid sequence around a highly conserved Asp was studied by site-directed mutagenesis. The results indicated that Lys253, Arg255, and Asp259 form a novel functional motif, K253XRXXXD259 (outside known motifs Exo I, II, and III), that is important not only for exonuclease activity but also for polymerizing activity, confirming functional interdependence between the polymerase and exonuclease domains. The short loop region, K253G254R255, probably contributes to binding to DNA substrates. Moreover, the negative charge and the side-chain length of D259 might play a supporting role in coordinating the conserved Mg2+ to the correct position at the active center in the exonuclease domain.

Molecular basis for the subunit assembly of the primase from an archaeon Pyrococcus horikoshii

Archaeal/eukaryotic primases form a heterodimer consisting of a small catalytic subunit (PriS) and a large subunit (PriL). The heterodimer complex synthesizes primer oligoribonucleotides that are required for chromosomal replication. Here, we describe crystallographic and biochemical studies of the N‐terminal domain (NTD) of PriL (PriLNTD; residues 1–222) that bind to PriS from a hyperthermophilic archaeon, Pyrococcus horikoshii, at 2.9 Å resolution. The PriLNTD structure consists of two subdomains, the helix‐bundle and twisted‐strand domains. The latter is structurally flexible, and is expected to contain a PriS interaction site. Pull‐down and surface plasmon resonance analyses of structure‐based deletion and alanine scanning mutants showed that the conserved hydrophobic Tyr155‐Tyr156‐Ile157 region near the flexible region is the PriS‐binding site, as the Y155A/Y156A/I157A mutation markedly reduces PriS binding, by 1000‐fold. These findings and a structural comparison with a previously reported PriLNTD–PriS complex suggest that the presented alternative conformations of the twisted‐strand domain facilitate the heterodimer assembly.

Clustering of OB-fold domains of the partner protease complexed with trimeric stomatin from Thermococcales.

The C-terminal soluble domain of stomatin operon partner protein (STOPP) of the hyperthermophilic archaeon Pyrococcus horikoshii has an oligonucleotide binding-fold (OB-fold). STOPP lacks the conserved surface residues necessary for binding to DNA/RNA. A tryptophan (W) residue is conserved instead at the molecular surface. Solvent-accessible W residues are often found at interfaces of protein-protein complexes, which suggested the possibility of self-assembling of STOPP. Protein-protein interactions among the C-terminal soluble domains of STOPP PH1510 (1510-C) were then analyzed by chemical linking and blue native polyacrylamide gel electrophoresis (BN-PAGE) methods. These results suggest that the soluble domains of STOPP could assemble into homo-oligomers. Since hexameric subcomplex I from archaeal proteasome consists of coiled-coil segments and OB-fold domains, molecular modeling of 1510-C was performed using hexameric subcomplex I as a template. Although 1510-C is a comparatively small polypeptide consisting of approximately 60 residues, numerous salt bridges and hydrophobic interactions were observed in the predicted hexamer of 1510-C, suggesting the stability of the homo-oligomeric structure. This oligomeric property of STOPP may be favorable for triplicate proteolysis of the trimer of prokaryotic stomatin.

Serial intermediates with a 1 nt 3′-flap and 5′ variable-length flaps are formed by cooperative functioning of Pyrococcus horikoshii FEN-1 with either B or D DNA polymerases

Flap endonuclease-1 (FEN-1) plays important roles with DNA polymerases in DNA replication, repair and recombination. FEN-1 activity is elevated by the presence of a 1 nucleotide expansion at the 3′ end in the upstream primer of substrates called “structures with a 1 nt 3′-flap”, which appear to be the most preferable substrates for FEN-1; however, it is unclear how such substrates are generated in vivo. Here, we show that substrate production occurred by the cooperative function of FEN-1(phFEN-1) and Pyrococcus horikoshii DNA polymerase B (phPol B) or D (phPol D). Using various substrates, the activities of several phFEN-1 F79 mutants were compared with those of the wild type. Analysis of the activity profiles of these mutants led us to discriminate “structures with a 1 nt 3′-flap” from substrates with a 3′ -projection longer than 2 nt or from those without a 3′-projection. When phFEN-1 processed a gap substrate with phPol B or phPol D, “structures with a 1 nt 3′-flap” were assumed the reaction intermediates. Furthermore, the phFEN-1 cleavage products with phPol B or D were from 1mer to 7mer, corresponding to the sizes of the strand-displacement products of these polymerases. This suggests that a series of 1 nt 3′-flap with 5′-variable length-flap configurations were generated as transient intermediates, in which the length of the 5′-flaps depended on the displacement distance of the downstream strand by phPol B or D. Therefore, phFEN-1 might act successively on displaced 5′-variable flaps.

Domain structures and inter-domain interactions defining the holoenzyme architecture of archaeal d-family DNA polymerase.

Archaea-specific D-family DNA polymerase (PolD) forms a dimeric heterodimer consisting of two large polymerase subunits and two small exonuclease subunits. According to the protein-protein interactions identified among the domains of large and small subunits of PolD, a symmetrical model for the domain topology of the PolD holoenzyme is proposed. The experimental evidence supports various aspects of the model. The conserved amphipathic nature of the N-terminal putative α-helix of the large subunit plays a key role in the homodimeric assembly and the self-cyclization of the large subunit and is deeply involved in the archaeal PolD stability and activity. We also discuss the evolutional transformation from archaeal D-family to eukaryotic B-family polymerase on the basis of the structural information.