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Glycosidases have long been used for the synthesis of glycosides by transglycosylation reactions. Especially glycosidases from hyperthermophilic bacteria are useful for reactions under extreme reaction conditions, e.g., in the presence of organic solvents. We herein report the facile enzymatic synthesis and purification of 2-(β-galactosyl)-ethyl methacrylate (Gal-EMA) with the recombinant hyperthermostable glycosidase from Pyrococcus woesei in high yields. Optimized reaction conditions resulted in gram-scale synthesis of the galactosylated monomer with 88% transglycosylation yield. The product Gal-EMA was characterized by high-performance liquid chromatography-electrospray ionization-mass spectrometry (HPLC-ESI-MS), nuclear magnetic resonance (NMR) spectroscopy, and infrared (IR) spectroscopy. Gal-EMA was utilized to synthesize sugar-functionalized acrylate polymers with defined amounts of incorporated galactose (0-100%). Analysis of the binding affinity of the lectin RCA 120 from Ricinus communis to the glycopolymers using an enzyme-linked lectin assay (ELLA) revealed K D values between 0.24 and 6.2 nM, depending on the amount of incorporated Gal-EMA. The potential of Gal-EMA for the synthesis of acrylate-functionalized glycan oligomers was demonstrated by sequential elongation of the terminal galactose by two glycosyltransferases, resulting in the terminal glycan N -acetyllactosamine (LacNAc) epitope. In conclusion, the enzymatic synthesis of Gal-EMA opens new routes to a series of novel monomeric building blocks for the synthesis of glycan-functionalized polyacrylates.

Pullulanase is a commonly used debranching enzyme in the starch processing industry. Because the starch liquefaction process requires high temperature, a thermostable pullulanase is desired. Here, a novel hyperthermostable type II pullulanase gene ( pul PY ) was cloned from Pyrococcus yayanosii CH1, isolated from a deep-sea hydrothermal site. Pul PY was optimally active at pH 6.6 and 95 °C, retaining more than 50% activity after incubation at 95 °C for 10 h. The thermostability was significantly higher than those of most pullulanases reported previously. To further improve its activity and thermostability, the N-terminal and C-terminal domains of Pul PY were truncated. The optimum temperature of the combined truncation mutant Δ28N + Δ791C increased to 100 °C with a specific activity of 32.18 U/mg, which was six times higher than that of wild-type Pul PY . Pul PY and the truncation mutant enzyme could realize the combined use of pullulanase with α-amylase during the starch liquefaction process to improve hydrolysis efficiency.

Transition metal catalysis is a powerful tool for chemical synthesis, a standard by which understanding of elementary chemical processes can be measured, and a source of awe for those who simply appreciate the difficulty of cleaving and forming chemical bonds. Each of these statements is amplified in cases where the transition metal catalyst controls the selectivity of a chemical reaction. Enantioselective catalysis is a challenging but well-established phenomenon, and regio- or site-selective catalysis is increasingly common. On the other hand, transition-metal-catalyzed reactions are typically conducted under highly optimized conditions. Rigorous exclusion of air and water is common, and it is taken for granted that only a single substrate (of a particular class) will be present in a reaction, a desired site selectivity can be achieved by installing a directing group, and undesired reactivity can be blocked with protecting groups. These are all reasonable synthetic strategies, but they also highlight limits to catalyst control. The utility of transition metal catalysis could be greatly expanded if catalysts possessed the ability to regulate which molecules they encounter and the relative orientation of those molecules. The rapid and widespread adoption of stoichiometric bioorthogonal reactions illustrates the utility of robust reactions that proceed with high selectivity and specificity under mild reaction conditions. Expanding this capability beyond preprogrammed substrate pairs via catalyst control could therefore have an enormous impact on molecular science. Many metalloenzymes exhibit this level of catalyst control, and directed evolution can be used to rapidly improve the catalytic properties of these systems. On the other hand, the range of reactions catalyzed by enzymes is limited relative to that developed by chemists. The possibility of imparting enzyme-like activity, selectivity, and evolvability to reactions catalyzed by synthetic transition metal complexes has inspired the creation of artificial metalloenzymes (ArMs). The increasing levels of catalyst control exhibited by ArMs developed to date suggest that these systems could constitute a powerful platform for bioorthogonal transition metal catalysis and for selective catalysis in general. This Account outlines the development of a new class of ArMs based on a prolyl oligopeptidase (POP) scaffold. Studies conducted on POP ArMs containing a covalently linked dirhodium cofactor have shown that POP can impart enantioselectivity to a range of dirhodium-catalyzed reactions, increase reaction rates, and improve the specificity for reaction of dirhodium carbene intermediates with targeted organic substrates over components of cell lysate, including bulk water. Several design features of these ArMs enabled their evolution via random mutagenesis, which revealed that mutations throughout the POP scaffold, beyond the second sphere of the dirhodium cofactor, were important for ArM activity and selectivity. While it was anticipated that the POP scaffold would be capable of encapsulating and thus controlling the selectivity of bulky cofactors, molecular dynamics studies also suggest that POP conformational dynamics plays a role in its unique efficacy. These advances in scaffold selection, bioconjugation, and evolution form the basis of our ongoing efforts to control transition metal reactivity using protein scaffolds with the goal of enabling unique synthetic capabilities, including bioorthogonal catalysis.

Protein N-glycosylation occurs in the three domains of life. Oligosaccharyltransferase (OST) transfers glycan to asparagine in the N-glycosylation sequon. The catalytic subunit of OST is called STT3 in eukaryotes, AglB in archaea, and PglB in eubacteria. The genome of a hyperthermophilic archaeon, Archaeoglobus fulgidus, encodes three AglB paralogs. Two of them are the shortest AglBs across all domains of life. We determined the crystal structure of the C-terminal globular domain of the smallest AglB to identify the minimal structural unit. The Archaeoglobus AglB lacked a β-barrel-like structure, which had been found in other AglB and PglB structures. In agreement, the deletion in a larger Pyrococcus AglB confirmed its dispensability for the activity. By contrast, the Archaeoglobus AglB contains a kinked helix bearing a conserved motif, called DK/MI motif. The lysine and isoleucine residues in the motif participate in the Ser/Thr recognition in the sequon. The Archaeoglobus AglB structure revealed that the kinked helix contained an unexpected insertion. A revised sequence alignment based on this finding identified a variant type of the DK motif with the insertion. A mutagenesis study of the Archaeoglobus AglB confirmed the contribution of this particular type of the DK motif to the activity. When taken together with our previous results, this study defined the classification of OST: one group consisting of eukaryotes and most archaea possesses the DK-type Ser/Thr pocket, and the other group consisting of eubacteria and the remaining archaea possesses the MI-type Ser/Thr pocket. This classification provides a useful framework for OST studies.

The minimal polypeptide supporting full methionyl-tRNA synthetase (MetRS) activity is composed of four domains: a catalytic Rossmann fold, a connective peptide, a KMSKS domain, and a C-terminal alpha helix bundle domain. The minimal MetRS behaves as a monomer. In several species, MetRS is a homodimer because of a C-terminal domain appended to the core polypeptide. Upon truncation of this C-terminal domain, subunits dissociate irreversibly. Here, the C-terminal domain of dimeric MetRS from Pyrococcus abyssi was isolated and studied. It displays nonspecific tRNA-binding properties and has a crystalline structure closely resembling that of Trbp111, a dimeric tRNA-binding protein found in many bacteria and archaea. The obtained 3D model was used to direct mutations against dimerization of Escherichia coli MetRS. Comparison of the resulting mutants to native and C-truncated MetRS shows that the presence of the appended C-domain improves tRNA(Met) binding affinity. However, dimer formation is required to evidence the gain in affinity.

A beta-mannosidase gene (PH0501) was identified in the Pyrococcus horikoshii genome and cloned and expressed in E. coli. The purified enzyme (BglB) was most specific for the hydrolysis of p-nitrophenyl-beta-D-mannopyranoside (pNP-Man) (Km: 0.44 mM) with a low turnover rate (kcat: 4.3 s(-1)). The beta-mannosidase has been classified as a member of family 1 of glycoside hydrolases. Sequence alignments and homology modeling showed an apparent conservation of its active site region with, remarkably, two unique active site residues, Gln77 and Asp206. These residues are an arginine and asparagine residue in all other known family 1 enzymes, which interact with the catalytic nucleophile and equatorial C2-hydroxyl group of substrates, respectively. The unique residues of P. horikoshii BglB were introduced in the highly active beta-glucosidase CelB of Pyrococcus furiosus and vice versa, yielding two single and one double mutant for each enzyme. In CelB, both substitutions R77Q and N206D increased the specificity for mannosides and reduced hydrolysis rates 10-fold. In contrast, BglB D206N showed 10-fold increased hydrolysis rates and 35-fold increased affinity for the hydrolysis of glucosides. In combination with inhibitor studies, it was concluded that the substituted residues participate in the ground-state binding of substrates with an equatorial C2-hydroxyl group, but contribute most to transition-state stabilization. The unique activity profile of BglB seems to be caused by an altered interaction between the enzyme and C2-hydroxyl of the substrate and a specifically increased affinity for mannose that results from Asp206.

Novel thymidine analogue triphosphates, which have an sp3-hybridized carbon at the C5 alpha-position with amino-linker arms, a methyl ester, or a carboxyl group at the C5 sidearm, were good substrates for primer-extension reactions by DNA polymerase from Pyrococcus kodakaraensis (KOD Dash DNA polymerase), yielding exclusively full-length products. The resulting modified DNA was further allowed to react with a functional molecule such as fluorescein isothiocyanate. By contrast, only truncated products were formed from the thymidine analogue substrate bearing the amino-linker arm or the negatively charged carboxyl group using Taq, Tth DNA polymerase, or DNA polymerase I from E. coli (Klenow fragment). The results indicate either that the thymidine analogue was not accepted by the enzymes, or that the polymerases could not extend the products, once the analogue had been incorporated, depending on the type of the analogue. A conventional thymidine analogue bearing an aminopropenyl group at the C5-position was accepted by all enzymes, among which KOD Dash DNA polymerase showed the highest activity for the polymerization with this analogue. Templates bearing the thymidine analogues in place of one thymidine residue were read by KOD Dash, Taq, Tth DNA polymerases, and the Klenow fragment giving the full-length product. KOD Dash DNA polymerase could expand structural diversities of substrates that can be used to prepare modified DNAs.

Bacterial pyrazinamidase (PZAase)/nicotinamidase converts pyrazinamide (PZA) to ammonia and pyrazinoic acid, which is active against Mycobacterium tuberculosis. Loss of PZAase activity is the major mechanism of pyrazinamide-resistance by M. tuberculosis. We have determined the crystal structure of the gene product of Pyrococcus horikoshii 999 (PH999), a PZAase, and its complex with zinc ion by X-ray crystallography. The overall fold of PH999 is similar to that of N-carbamoylsarcosine amidohydrolase (CSHase) of Arthrobacter sp. and YcaC of Escherichia coli, a protein with unknown physiological function. The active site of PH999 was identified by structural features that are also present in the active sites of CSHase and YcaC: a triad (D10, K94, and C133) and a cis-peptide (between V128 and A129). Surprisingly, a metal ion-binding site was revealed in the active site and subsequently confirmed by crystal structure of PH999 in complex with Zn(2+). The roles of the triad, cis-peptide, and metal ion in the catalysis are proposed. Because of extensive homology between PH999 and PZAase of M. tuberculosis (37% sequence identity), the structure of PH999 provides a structural basis for understanding PZA-resistance by M. tuberculosis harboring PZAase mutations.

The complexes TpWO2X react with sulfiding agents such as B2S3 or P4S10 to give the oxothio- and bis(thio)tungsten(VI) complexes TpWOSX (X = Cl(-)) and TpWS2X [X = Cl(-), S2PPh2(-); Tp = hydrotris(3,5-dimethylpyrazol-1-yl)borate]. The reaction of TpWS2Cl with (i) PPh3 in pyridine and (ii) dimethyl sulfoxide affords TpWOSCl in good overall yield. The chloro complexes undergo metathesis with alkali metal salts to yield species of the type TpWOSX and TpWS2X [X = OPh(-), SPh(-), SePh(-), (-)-mentholate]. The diamagnetic complexes exhibit NMR spectra indicative of C(1) (TpWOSX) or C(s) (TpWS2X) symmetry and IR spectra consistent with terminal oxo and thio ligation (nu(W=O), 940-925 cm(-1); nu(W=S) or nu(WS2), 495-475 cm(-1)). Crystals of (R,S)-TpWOS[(-)-mentholate] are monoclinic, space group P2(1), with a = 11.983(2) A, b = 18.100(3) A, c = 13.859(3) A, beta = 91.60(2) degrees, V = 3004.6(8) A(3), and Z = 4. Crystals of TpWS2(OPh)-CH2Cl2 are orthorhombic, space group Pbca, with a = 16.961(4) A, b = 33.098(7) A, c = 9.555(2) A, V = 5364(2) A(3), and Z = 8. The mononuclear, distorted-octahedral tungsten centers are coordinated by a tridentate Tp ligand, an alkoxy or aryloxy ligand, and two terminal chalcogenide ligands. The average W=O and W=S distances are 1.726(7) and 2.125(2) A, respectively, and the O=W=S and S=W=S angles 102.9(3) and 102.9(1) degrees, respectively. The tungsten and sulfur X-ray absorption spectra of TpWOSCl and TpWS2Cl are consistent with the presence of terminal pi-bonded thio ligands in both complexes. The thio complexes generally undergo a reversible one-electron reduction at potentials significantly more positive than their oxo analogues. The chemical, spectroscopic, and electrochemical properties of the complexes are heavily influenced by the presence of W=S pi frontier orbitals.

A simplified and rapid procedure for the determination of pyroglutamic acid in peptides was developed. The method involves the enzymatic cleavage of an N-terminal pyroglutamate residue using a thermostable pyroglutamate aminopeptidase and isocratic HPLC separation of the resulting enzymatic hydrolysate using a column switching technique. Pyroglutamate aminopeptidase from a thermophilic archaebacteria, Pyrococcus furiosus, cleaves N-terminal pyroglutamic acid residue independent of the molecular weight of the substrate. It cleaves more than 85% of pyroglutamate from peptides whose molecular weight ranges from 362.4 to 4599.4 Da. Thus, a new method is presented that quantitatively estimates N-terminal pyroglutamic acid residue in peptides.

Pyruvate ferredoxin oxidoreductase (POR) from the hyperthermophilic archaeon Pyrococcus furiosus (Pf) catalyzes the final oxidative step in carbohydrate fermentation in which pyruvate is oxidized to acetyl-CoA and CO2, coupled to the reduction of ferredoxin (Fd). POR is composed of two 'catalytic units' of molecular mass approximately 120 kDa. Each unit consists of four subunits, alpha beta gamma delta, with masses of approximately 44, 36, 20, and 12 kDa, respectively, and contains at least two [4Fe-4S] clusters. The precise mechanism of catalysis and the role of the individual subunits are not known. The gene encoding the delta-subunit of Pf POR has been expressed in E. coli, and the protein was purified after reconstitution with iron and sulfide. The reconstituted delta-subunit (recPOR-delta) is monomeric with a mass of 11 879 +/- 1.2 Da as determined by mass spectrometry, in agreement with that predicted from the gene sequence. Purified recPOR-delta contains 8 Fe mol/mol and remained intact when incubated at 85 degreesC for 2 h, as judged by its visible absorption properties. The reduced form of the protein exhibited an EPR spectrum characteristic of two, spin-spin interacting [4Fe-4S]1+ clusters. When compared with the EPR properties of the reduced holoenzyme, the latter was shown to contain a third [4Fe-4S]1+ cluster in addition to the two within the delta-subunit. The reduction potential of the two 4Fe clusters in isolated recPOR-delta (-403 +/- 8 mV at pH 8.0 and 24 degreesC) decreased linearly with temperature (-1.55 mV/ degreesC) up to 82 degreesC. RecPOR-delta replaced Pf Fd as an in vitro electron carrier for two oxidoreductases from Pf, POR and Fd:NADP oxidoreductase, and the POR holoenzyme displayed a higher apparent affinity for its own subunit (apparent Km = 1.0 microM at 80 degreesC) than for Fd (apparent Km = 4.4 microM). The molecular and spectroscopic properties and amino acid sequence of the isolated delta-subunit suggest that it evolved from an 8Fe-type Fd by the addition of approximately 40 residues at the N-terminus, and that this extension enabled it to interact with additional subunits within POR.

The ferredoxin (7.5 kDa) of the hyperthermophilic archaeon, Pyrococcus furiosus, contains a single [4Fe-4S]1+,2+ cluster that is coordinated by three Cys and one Asp residue rather than the expected four Cys. The role of this Asp residue was investigated using a series of mutants, D14X, where X = C, S, H, N, V, and Y, prepared by heterologous gene expression in Escherichia coli. While the recombinant form of the wild-type and the D14S and D14C mutants contained a [4Fe-4S]1+,2+ cluster, the D14V, D14H, D14Y, and D14N proteins contained a [3Fe-4S]0,+ center, as determined by visible spectroscopy and electrochemistry. The redox potentials (at pH 7.0, 23 degrees C) of the D14C and D14S mutants were decreased by 58 and 133 mV, respectively, compared to those of the wild-type 4Fe-ferredoxin (Em -368 mV), while those of the 3Fe-protein mutants (including the 3Fe-form of the D14S, generated by chemical oxidation) were between 15 and 118 mV more positive than that of wild-type 3Fe-form (obtained by chemical oxidation, Em -203 mV). The reduction potentials of all of the 3Fe-forms, except the D14S mutant, showed a pH response over the range 3.0-10.0 with a pK of 3.3-4.7, and this was assigned to cluster protonation. The D14H mutant and the wild-type 3Fe-proteins showed an additional pK (both at 5.9) assumed to arise from protonation of the amino acid side chain. With the 4Fe-proteins, there was no dramatic change in the potentials of the wild-type or D14C form, while the pH response of the D14S mutant (pK 4.75) was ascribed to protonation of the serinate. While the ferredoxin variants exhibited a range of thermal stabilities (measured at 80 degrees C, pH 2.5), none of them showed any temperature-dependent transitions (0-80 degrees C) in their reduction potentials, and there was no correlation between the calculated DeltaS degrees' values and the absorbance maximum, reduction potential, or hydrophobicity of residue 14. In contrast, there was a linear correlation between the DeltaH degrees' value and reduction potential. Kinetic analyses were carried out at 80 degrees C using the ferredoxin as either an electron acceptor to pyruvate oxidoreductase (POR) or as an electron donor to ferredoxin:NADP oxidoreductase (FNOR, both from P. furiosus). The data showed that the reduction potential of the ferredoxin, rather than cluster type or the nature of the residue at position 14, appears to be the predominant factor in determining efficiency of electron transfer in both systems. However, compared to all the variants, the reduction potential of WT Fd makes it the most appropriate protein to both accept electrons from POR and donate them to FNOR.

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.