NAD+-dependent RNA terminal 2' and 3' phosphomonoesterase activity of a subset of Tpt1 enzymes.

The enzyme Tpt1 removes the 2'-PO4 at the splice junction generated by fungal tRNA ligase; it does so via a two-step reaction in which (i) the internal RNA 2'-PO4 attacks NAD+ to form an RNA-2'-phospho-ADP-ribosyl intermediate; and (ii) transesterification of the ribose O2″ to the 2'-phosphodiester yields 2'-OH RNA and ADP-ribose-1″,2″-cyclic phosphate products. The role that Tpt1 enzymes play in taxa that have no fungal-type RNA ligase remains obscure. An attractive prospect is that Tpt1 enzymes might catalyze reactions other than internal RNA 2'-PO4 removal, via their unique NAD+-dependent transferase mechanism. This study extends the repertoire of the Tpt1 enzyme family to include the NAD+-dependent conversion of RNA terminal 2' and 3' monophosphate ends to 2'-OH and 3'-OH ends, respectively. The salient finding is that different Tpt1 enzymes vary in their capacity and positional specificity for terminal phosphate removal. Clostridium thermocellum and Aeropyrum pernix Tpt1 proteins are active on 2'-PO4 and 3'-PO4 ends, with a 2.4- to 2.6-fold kinetic preference for the 2'-PO4 The accumulation of a terminal 3'-phospho-ADP-ribosylated RNA intermediate during the 3'-phosphotransferase reaction suggests that the geometry of the 3'-p-ADPR adduct is not optimal for the ensuing transesterification step. Chaetomium thermophilum Tpt1 acts specifically on a terminal 2'-PO4 end and not with a 3'-PO4 In contrast, Runella slithyformis Tpt1 and human Tpt1 are ineffective in removing either a 2'-PO4 or 3'-PO4 end.

Total Synthesis and Structure Confirmation of trans-Anhydromevalonate-5-phosphate, a Key Biosynthetic Intermediate of the Archaeal Mevalonate Pathway.

The mevalonate pathway is an upstream terpenoid biosynthetic route of terpenoids for providing the two five-carbon units, dimethylallyl diphosphate, and isopentenyl diphosphate. Recently, trans-anhydromevalonate-5-phosphate (tAHMP) was isolated as a new biosynthetic intermediate of the archaeal mevalonate pathway. In this study, we would like to report the first synthesis of tAHMP and its enzymatic transformation using one of the key enzymes, mevalonate-5-phosphate dehydratase from a hyperthermophilic archaeon, Aeropyrum pernix. Starting from methyl tetrolate, a Cu-catalyzed allylation provided an E-trisubstituted olefin in a stereoselective manner. The resulting E-olefin was transformed to tAHMP by cleavage of the olefin and phosphorylation. The structure of the synthetic tAHMP was unambiguously determined by NOESY analysis.

Insights into the Maturation of Pernisine, a Subtilisin-Like Protease from the Hyperthermophilic Archaeon Aeropyrum pernix.

Pernisine is a subtilisin-like protease that was originally identified in the hyperthermophilic archaeon Aeropyrum pernix, which lives in extreme marine environments. Pernisine shows exceptional stability and activity due to the high-temperature conditions experienced by A. pernix Pernisine is of interest for industrial purposes, as it is one of the few proteases that has demonstrated prion-degrading activity. Like other extracellular subtilisins, pernisine is synthesized in its inactive pro-form (pro-pernisine), which needs to undergo maturation to become proteolytically active. The maturation processes of mesophilic subtilisins have been investigated in detail; however, less is known about the maturation of their thermophilic homologs, such as pernisine. Here, we show that the structure of pro-pernisine is disordered in the absence of Ca2+ ions. In contrast to the mesophilic subtilisins, pro-pernisine requires Ca2+ ions to adopt the conformation suitable for its subsequent maturation. In addition to several Ca2+-binding sites that have been conserved from the thermostable Tk-subtilisin, pernisine has an additional insertion sequence with a Ca2+-binding motif. We demonstrate the importance of this insertion for efficient folding and stabilization of pernisine during its maturation. Moreover, analysis of the pernisine propeptide explains the high-temperature requirement for pro-pernisine maturation. Of note, the propeptide inhibits the pernisine catalytic domain more potently at high temperatures. After dissociation, the propeptide is destabilized at high temperatures only, which leads to its degradation and finally to pernisine activation. Our data provide new insights into and understanding of the thermostable subtilisin autoactivation mechanism.IMPORTANCE Enzymes from thermophilic organisms are of particular importance for use in industrial applications, due to their exceptional stability and activity. Pernisine, from the hyperthermophilic archaeon Aeropyrum pernix, is a proteolytic enzyme that can degrade infective prion proteins and thus has a potential use for disinfection of prion-contaminated surfaces. Like other subtilisin-like proteases, pernisine needs to mature through an autocatalytic process to become an active protease. In the present study, we address the maturation of pernisine and show that the process is regulated specifically at high temperatures by the propeptide. Furthermore, we demonstrate the importance of a unique Ca2+-binding insertion for stabilization of mature pernisine. Our results provide a novel understanding of thermostable subtilisin autoactivation, which might advance the development of these enzymes for commercial use.

NAD+-dependent synthesis of a 5'-phospho-ADP-ribosylated RNA/DNA cap by RNA 2'-phosphotransferase Tpt1.

RNA 2'-phosphotransferase Tpt1 converts an internal RNA 2'-monophosphate to a 2'-OH via a two-step NAD+-dependent mechanism in which: (i) the 2'-phosphate attacks the C1″ of NAD+ to expel nicotinamide and form a 2'-phospho-ADP-ribosylated RNA intermediate; and (ii) the ADP-ribose O2″ attacks the phosphate of the RNA 2'-phospho-ADPR intermediate to expel the RNA 2'-OH and generate ADP-ribose 1″-2″ cyclic phosphate. Tpt1 is an essential component of the fungal tRNA splicing pathway that generates a unique 2'-PO4, 3'-5' phosphodiester splice junction during tRNA ligation. The wide distribution of Tpt1 enzymes in taxa that have no fungal-type RNA ligase raises the prospect that Tpt1 might catalyze reactions other than RNA 2'-phosphate removal. A survey of Tpt1 enzymes from diverse sources reveals that whereas all of the Tpt1 enzymes are capable of NAD+-dependent conversion of an internal RNA 2'-PO4 to a 2'-OH (the canonical Tpt1 reaction), a subset of Tpt1 enzymes also catalyzed NAD+-dependent ADP-ribosylation of an RNA or DNA 5'-monophosphate terminus. Aeropyrum pernix Tpt1 (ApeTpt1) is particularly adept in this respect. One-step synthesis of a 5'-phospho-ADP-ribosylated cap structure by ApeTpt1 (with no subsequent 5'-phosphotransferase step) extends the repertoire of the Tpt1 enzyme family and the catalogue of ADP-ribosylation reactions involving nucleic acid acceptors.

Extracellular production of the engineered thermostable protease pernisine from Aeropyrum pernix K1 in Streptomyces rimosus.

BACKGROUND: The thermostable serine protease pernisine originates from the hyperthermophilic Archaeaon Aeropyrum pernix and has valuable industrial applications. Due to its properties, A. pernix cannot be cultivated in standard industrial fermentation facilities. Furthermore, pernisine is a demanding target for heterologous expression in mesophilic heterologous hosts due to the relatively complex processing step involved in its activation. RESULTS: We achieved production of active extracellular pernisine in a Streptomyces rimosus host through heterologous expression of the codon-optimised gene by applying step-by-step protein engineering approaches. To ensure secretion of fully active enzyme, the srT signal sequence from the S. rimosus protease was fused to pernisine. To promote correct processing and folding of pernisine, the srT functional cleavage site motif was fused directly to the core pernisine sequence, this way omitting the proregion. Comparative biochemical analysis of the wild-type and recombinant pernisine confirmed that the enzyme produced by S. rimosus retained all of the desired properties of native pernisine. Importantly, the recombinant pernisine also degraded cellular and infectious bovine prion proteins, which is one of the particular applications of this protease. CONCLUSION: Functional pernisine that retains all of the advantageous properties of the native enzyme from the thermophilic host was successfully produced in a S. rimosus heterologous host. Importantly, we achieved extracellular production of active pernisine, which significantly simplifies further downstream procedures and also omits the need for any pre-processing step for its activation. We demonstrate that S. rimosus can be used as an attractive host for industrial production of recombinant proteins that originate from thermophilic organisms.

Heme A synthase in bacteria depends on one pair of cysteinyls for activity.

Heme A is a prosthetic group unique for cytochrome a-type respiratory oxidases in mammals, plants and many microorganisms. The poorly understood integral membrane protein heme A synthase catalyzes the synthesis of heme A from heme O. In bacteria, but not in mitochondria, this enzyme contains one or two pairs of cysteine residues that are present in predicted hydrophilic polypeptide loops on the extracytoplasmic side of the membrane. We used heme A synthase from the eubacterium Bacillus subtilis and the hyperthermophilic archeon Aeropyrum pernix to investigate the functional role of these cysteine residues. Results with B. subtilis amino acid substituted proteins indicated the pair of cysteine residues in the loop connecting transmembrane segments I and II as being essential for catalysis but not required for binding of the enzyme substrate, heme O. Experiments with isolated A. pernix and B. subtilis heme A synthase demonstrated that a disulfide bond can form between the cysteine residues in the same loop and also between loops showing close proximity of the two loops in the folded enzyme protein. Based on the findings, we propose a classification scheme for the four discrete types of heme A synthase found so far in different organisms and propose that essential cysteinyls mediate transfer of reducing equivalents required for the oxygen-dependent catalysis of heme A synthesis from heme O.

Role of F225 in O-phosphoserine sulfhydrylase from Aeropyrum pernix K1.

O-Phosphoserine sulfhydrylase (OPSS) synthesizes cysteine from O-phospho-L-serine (OPS) and sulfide. We have determined the three-dimensional structures of OPSS from hyperthermophilic archaeon Aeropyrum pernix K1 (ApOPSS) in complex with aminoacrylate intermediate (AA) formed from pyridoxal 5'-phosphate with OPS or in complex with cysteine and compared them with that of ApOPSS. We found an orientational change of F225 at the active-site entrance and constructed an F225A mutant to examine its activities and AA stability and clarify the role of F225 in ApOPSS. The OPS and O-acetyl-L-serine (OAS) sulfhydrylase activities of the F225A mutant decreased by 4.2- and 15-fold compared to those of the wild-type (wt) ApOPSS, respectively. The ability of OPS and OAS to form AA also decreased by 12- and 27-fold, respectively. AA was less stable in the F225A mutant than in the wt ApOPSS. Simulated docking showed that leaving groups, such as phosphate and acetate, were oriented to the inside of the active site in the F225A mutant, whereas they were oriented to the entrance in the wt ApOPSS. These results suggest that F225 in ApOPSS plays important roles in maintaining the hydrophobic environment of AA from solvent water and in controlling the orientation of leaving groups.

Ultrasound-Assisted Enantioselective Esterification of Ibuprofen Catalyzed by a Flower-Like Nanobioreactor.

A flower-like nanobioreactor was prepared for resolution of ibuprofen in organic solvents. Ultrasound irradiation has been used to improve the enzyme performance of APE1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1) in the enantioselective esterification. Under optimum reaction conditions (ultrasound power, 225 W; temperature, 45 °C; water activity, 0.21), the immobilized APE1547 showed an excellent catalytic performance (enzyme activity, 13.26 µmol/h/mg; E value, 147.1). After ten repeated reaction batches, the nanobioreactor retained almost 100% of its initial enzyme activity and enantioselectivity. These results indicated that the combination of the immobilization method and ultrasound irradiation can enhance the enzyme performance dramatically.

Catalytically distinct states captured in a crystal lattice: the substrate-bound and scavenger states of acylaminoacyl peptidase and their implications for functionality.

Acylaminoacyl peptidase (AAP) is an oligopeptidase that only cleaves short peptides or protein segments. In the case of AAP from Aeropyrum pernix (ApAAP), previous studies have led to a model in which the clamshell-like opening and closing of the enzyme provides the means of substrate-size selection. The closed form of the enzyme is catalytically active, while opening deactivates the catalytic triad. The crystallographic results presented here show that the open form of ApAAP is indeed functionally disabled. The obtained crystal structures also reveal that the closed form is penetrable to small ligands: inhibitor added to the pre-formed crystal was able to reach the active site of the rigidified protein, which is only possible through the narrow channel of the propeller domain. Molecular-dynamics simulations investigating the structure of the complexes formed with longer peptide substrates showed that their binding within the large crevice of the closed form of ApAAP leaves the enzyme structure unperturbed; however, their accessing the binding site seems more probable when assisted by opening of the enzyme. Thus, the open form of ApAAP corresponds to a scavenger of possible substrates, the actual cleavage of which only takes place if the enzyme is able to re-close.

Thermostability and reactivity in organic solvent of O-phospho-L-serine sulfhydrylase from hyperthermophilic archaeon Aeropyrum pernix K1.

O-phospho-l-serine sulfhydrylase (OPSS) from archaeon Aeropyrum pernix K1 is able to synthesize l-cysteine even at 80 °C. In this article, we compared thermal stability and reactivity in organic solvent of OPSS with those of O-acetyl-l-serine sulfhydrylase B (OASS-B) from Escherichia coli. As a result, the thermostability of OPSS was much higher than that of OASS-B. Moreover, the activity of OPSS increased in the reaction mixture containing the organic solvent, such as N, N'-dimethyl formamide and 1,4-dioxane, whereas that of OASS-B gradually decreased as the content of organic solvent increased. From the crystal structural analysis, the intramolecular electrostatic interactions of N-terminal domain in OPSS seemed to be correlated with the tolerance of OPSS to high temperature and organic solvent. These results indicate that OPSS is more superior to OASS-B for the industrial production of l-cysteine and unnatural amino acids that are useful pharmaceuticals in the presence of organic solvent.

Yeast mitochondrial threonyl-tRNA synthetase recognizes tRNA isoacceptors by distinct mechanisms and promotes CUN codon reassignment.

Aminoacyl-tRNA synthetases (aaRSs) ensure faithful translation of mRNA into protein by coupling an amino acid to a set of tRNAs with conserved anticodon sequences. Here, we show that in mitochondria of Saccharomyces cerevisiae, a single aaRS (MST1) recognizes and aminoacylates two natural tRNAs that contain anticodon loops of different size and sequence. Besides a regular tRNA(2Thr) with a threonine (Thr) anticodon, MST1 also recognizes an unusual tRNA(1Thr), which contains an enlarged anticodon loop and an anticodon triplet that reassigns the CUN codons from leucine to threonine. Our data show that MST1 recognizes the anticodon loop in both tRNAs, but employs distinct recognition mechanisms. The size but not the sequence of the anticodon loop is critical for tRNA(1Thr) recognition, whereas the anticodon sequence is essential for aminoacylation of tRNA(2Thr). The crystal structure of MST1 reveals that, while lacking the N-terminal editing domain, the enzyme closely resembles the bacterial threonyl-tRNA synthetase (ThrRS). A detailed structural comparison with Escherichia coli ThrRS, which is unable to aminoacylate tRNA(1Thr), reveals differences in the anticodon-binding domain that probably allow recognition of the distinct anticodon loops. Finally, our mutational and modeling analyses identify the structural elements in MST1 (e.g., helix α11) that define tRNA selectivity. Thus, MTS1 exemplifies that a single aaRS can recognize completely divergent anticodon loops of natural isoacceptor tRNAs and that in doing so it facilitates the reassignment of the genetic code in yeast mitochondria.

Enzymatic resolution of ibuprofen in an organic solvent under ultrasound irradiation.

Ultrasound has been successfully adopted to improve the biocatalytic properties of APE1547 (a novel esterase from the archaeon Aeropyrum pernix K1) in the resolution of ibuprofen. After optimizing the conditions (ultrasound power, 200 W; temperature, 35 °C), the best biocatalytic performance of APE1547 (enzyme activity, 5.39 µmol/H/mg; E value, 130.8) was obtained. Compared with the conventional reaction in an orbital shaker, the enzyme activity was significantly enhanced about 90-fold, and the enantioselectivity was enhanced about fourfold after an ultrasound. The results of scanning electron microscopy clearly indicated that the activation effect of ultrasound on APE1547 originated mainly in the morphological change of the enzyme powder. Both lower particle size and conformational change of APE1547 under ultrasound might be helpful to enhance the enantioselectivity. In addition, APE1547 kept its best performance under the low-power ultrasound for at least five reaction cycles.

Crystal structure of novel dye-linked L-proline dehydrogenase from hyperthermophilic archaeon Aeropyrum pernix.

Two types of dye-linked L-proline dehydrogenase (PDH1, α4ß4-type hetero-octamer, and PDH2, αßγδ-type heterotetramer) have been identified so far in hyperthermophilic archaea. Here, we report the crystal structure of a third type of L-proline dehydrogenase, found in the aerobic hyperthermophilic archaeon Aeropyrum pernix, whose structure (homodimer) is much simpler than those of previously studied L-proline dehydrogenases. The structure was determined at a resolution of 1.92 Å. The asymmetric unit contained one subunit, and a crystallographic 2-fold axis generated the functional dimer. The overall fold of the subunit showed similarity to that of the PDH1 ß-subunit, which is responsible for catalyzing L-proline dehydrogenation. However, the situation at the subunit-subunit interface of the A. pernix enzyme was totally different from that in PDH1. The presence of additional surface elements in the A. pernix enzyme contributes to a unique dimer association. Moreover, the C-terminal Leu(428), which is provided by a tail extending from the FAD-binding domain, shielded the active site, and an L-proline molecule was entrapped within the active site cavity. The K(m) value of a Leu(428) deletion mutant for L-proline was about 800 times larger than the K(m) value of the wild-type enzyme, although the k(cat) values did not differ much between the two enzymes. This suggests the C-terminal Leu(428) is not directly involved in catalysis, but it is essential for maintaining a high affinity for the substrate. This is the first description of an LPDH structure with L-proline bound, and it provides new insight into the substrate binding of LPDH.

Substrate specificity of undecaprenyl diphosphate synthase from the hyperthermophilic archaeon Aeropyrum pernix.

Cis-prenyltransferase from a hyperthermophilic archaeon Aeropyrum pernix was expressed in Escherichia coli and purified for characterization. Properties such as substrate specificity, product chain-length, thermal stability and cofactor requirement were investigated using the recombinant enzyme. In particular, the substrate specificity of the enzyme attracts interest because only dimethylallyl diphosphate and geranylfarnesyl diphosphate, both of which are unusual substrates for known cis-prenyltransferases, are likely available as an allylic primer substrate in A. pernix. From the enzymatic study, the archaeal enzyme was shown to be undecaprenyl diphosphate synthase that has anomalous substrate specificity, which results in a preference for geranylfarnesyl diphosphate. This means that the product of the enzyme, which is probably used as the precursor of the glycosyl carrier lipid, would have an undiscovered structure.

Cleavage of intron from the standard or non-standard position of the precursor tRNA by the splicing endonuclease of Aeropyrum pernix, a hyper-thermophilic Crenarchaeon, involves a novel RNA recognition site in the Crenarchaea specific loop.

In Crenarchaea, several tRNA genes are predicted to express precursor-tRNAs (pre-tRNAs) with canonical or non-canonical introns at various positions. We initially focused on the tRNA(Thr) species of hyperthermophilic crenarchaeon, Aeropyrum pernix (APE) and found that in the living APE cells three tRNA(Thr) species were transcribed and subsequently matured to functional tRNAs. During maturation, introns in two of them were cleaved from standard and non-standard positions. Biochemical studies revealed that the APE splicing endonuclease (APE-EndA) removed both types of introns, including the non-canonical introns, without any nucleotide modification. To clarify the underlying reasons for broad substrate specificity of APE-EndA, we determined the crystal structure of wild-type APE-EndA and subsequently compared its structure with that of Archaeaoglobus fulgidus (AFU)-EndA, which has narrow substrate specificity. Remarkably, structural comparison revealed that APE-EndA possesses a Crenarchaea specific loop (CSL). Introduction of CSL into AFU-EndA enhanced its intron-cleaving activity irrespective of the position or motif of the intron. Thus, our biochemical and crystallographic analyses of the chimera-EndA demonstrated that the CSL is responsible for the broad substrate specificity of APE-EndA. Furthermore, mutagenesis studies revealed that Lys44 in CSL functions as the RNA recognition site.

Enantioselective esterification of ibuprofen under microwave irradiation.

Enantioselective esterification of ibuprofen has been successfully carried out in an organic solvent catalyzed by recombinant APE 1547 (a thermophilic esterase from the archaeon Aeropyrum pernix K1). Here we used microwave irradiation (MW) as the mode of heating to improve the enzyme performance. Under the optimum conditions, the enzyme activity of APE 1547 was 4.16 µmol/mg/h and the enantioselectivity (E value) was 52.9. Compared with conventional heating, the enzyme activity and the enantioselectivity were increased about 21.9-fold and 1.4-fold, respectively. The results also indicated that APE 1547 can maintain 95% of its activity even after being used five times, suggesting that the enzyme is stable under low power MW conditions.

Structure and catalysis of acylaminoacyl peptidase: closed and open subunits of a dimer oligopeptidase.

Acylaminoacyl peptidase from Aeropyrum pernix is a homodimer that belongs to the prolyl oligopeptidase family. The monomer subunit is composed of one hydrolase and one propeller domain. Previous crystal structure determinations revealed that the propeller domain obstructed the access of substrate to the active site of both subunits. Here we investigated the structure and the kinetics of two mutant enzymes in which the aspartic acid of the catalytic triad was changed to alanine or asparagine. Using different substrates, we have determined the pH dependence of specificity rate constants, the rate-limiting step of catalysis, and the binding of substrates and inhibitors. The catalysis considerably depended both on the kind of mutation and on the nature of the substrate. The results were interpreted in terms of alterations in the position of the catalytic histidine side chain as demonstrated with crystal structure determination of the native and two mutant structures (D524N and D524A). Unexpectedly, in the homodimeric structures, only one subunit displayed the closed form of the enzyme. The other subunit exhibited an open gate to the catalytic site, thus revealing the structural basis that controls the oligopeptidase activity. The open form of the native enzyme displayed the catalytic triad in a distorted, inactive state. The mutations affected the closed, active form of the enzyme, disrupting its catalytic triad. We concluded that the two forms are at equilibrium and the substrates bind by the conformational selection mechanism.

Two-step efficient synthesis of 5-methyluridine via two thermostable nucleoside phosphorylase from Aeropyrum pernix.

5-Methyluridine has been synthesized in high yield using guanosine and thymine as starting materials in the presence of highly thermostable recombinant purine nucleoside phosphorylase (PNP) and uridine phosphorylase (UP) obtained from hyperthermophilic aerobic crenarchaeon Aeropyrum pernix. Key reaction parameters such as pH, temperature, concentration of buffer and substrates were investigated. At the optimal conditions, 5-methyluridine was achieved in yield 85% with a guanosine conversion of 96% in 10ml scale. The process can be performed at high temperature, which will highly increase the solubility of substrates, therefore, this process is suitable for the industry application.

Cloning, expression, purification, crystallization and preliminary X-ray crystallographic analyses of threonyl-tRNA synthetase editing domain from Aeropyrum pernix.

The proofreading function of aminoacyl-tRNA synthetases is crucial in maintaining the fidelity of protein synthesis. Most archaeal threonyl-tRNA synthetases (ThrRSs) possess a unique proofreading domain unrelated to their eukaryotic/bacterial counterpart. The crystal structure of this domain from the archaeon Pyrococcus abysii in complex with its cognate and noncognate substrate analogues had given insights into its catalytic and discriminatory mechanisms. To probe further into the mechanistic and evolutionary aspects of this domain, work has been extended to another archaeon Aeropyrum pernix. The organism possesses two proteins corresponding to threonyl-tRNA synthetase, i.e. ThrRS1 and ThrRS2, encoded by two different genes, thrS1 and thrS2, respectively. ThrRS1 is responsible for aminoacylation and ThrRS2 for proofreading activity. Here the purification, crystallization and preliminary X-ray crystallographic investigation of the N-terminal proofreading domain of ThrRS2 from A. pernix is reported. The crystals belong to either the P4(1)2(1)2 or P4(3)2(1)2 space group and consist of one monomer per asymmetric unit.

Structure and characterization of the 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase from Aeropyrum pernix.

The first enzyme in the shikimic acid biosynthetic pathway, 3-deoxy-D-arabino-heptulosonate 7-phosphate synthase (DAH7PS), varies significantly in size and complexity in the bacteria and plants that express it. The DAH7PS from the archaebacterium Aeropyrum pernix (DAH7PS(Ap)) is among the smallest and least complex of the DAH7PS enzymes, leading to the hypothesis that DAH7PS(Ap) would not be subject to feedback regulation by shikimic acid pathway products. We overexpressed DAH7PS(Ap) in Escherichia coli, purified it, and characterized its enzymatic activity. We then solved its X-ray crystal structure with a divalent manganese ion and phosphoenolpyruvate bound (PDB ID: 1VS1). DAH7PS(Ap) is a homodimeric metalloenzyme in solution. Its enzymatic activity increases dramatically above 60 °C, with optimum activity at 95 °C. Its pH optimum at 60 °C is 5.7. DAH7PS(Ap) follows Michaelis-Menten kinetics at 60 °C, with a K(M) for erythrose 4-phosphate of 280 µM, a K(M) for phosphoenolpyruvate of 891 µM, and a k(cat) of 1.0 s(-1). None of the downstream products of the shikimate biosynthetic pathway we tested inhibited the activity of DAH7PS(Ap). The structure of DAH7PS(Ap) is similar to the structures of DAH7PS from Thermatoga maritima (PDB ID: 3PG8) and Pyrococcus furiosus (PDB ID: 1ZCO), and is consistent with its designation as an unregulated DAH7PS.