Investigation on the effects of three X-->histidine replacements on thermostability of alpha-amylase from Bacillus amyloliquefaciens

Bacillus licheniformis alpha-amylase (BLA), a thermophilic counterpart of Bacillus amyloliquefaciens alpha-amylase (BAA), is an appropriate model for the design of stabilizing mutations in BAA. BLA has 10 more histidines than BAA. Considering this prominent difference, in the present study, three out of these positions (I34, Q67, and P407; located in the thermostability determinant 1 region and Ca-III binding site of BAA) were replaced with histidine in BAA, using the site-directed mutagenesis technique. The results showed that the thermostability of P407H and Q67H mutants had increased, but no significant changes were observed in their kinetic parameters compared to that of the wild type. I34H replacement resulted in complete loss of enzyme activity. Moreover, fluorescence and circular dichroism data indicated a more rigid structure for the P407H variant compared with that of the wild-type BAA. However, the flexibility of Q67H and I34H mutants increased in comparison with that of wild-type enzyme.     

The structural roles of conserved Pro196, Pro197 and His199 in the mechanism of thymidylate synthase

We generated replacement sets for three highly conserved residues, Pro196, Pro197 and His199, that flank the catalytic nucleophile, Cys198. Pro196 and Pro197 have restricted mobility that could be important for the structural transitions known to be essential for activity. To test this hypothesis we obtained and characterized 13 amino acid substitutions for Pro196, 14 for Pro197 and 14 for His199. All of the Pro196 and Pro197 variants, except P197R, and four of the His199 variants complemented TS-deficient Escherichia coli cells, indicating they had at least 1% of wild-type activity. For all His199 mutations, k(cat)/K(m) for substrate and cofactor decreased more than 40-fold, suggesting that the conserved hydrogen bond network co-ordinated by His199 is important for catalysis. Pro196 can be substituted with small hydrophilic residues with little loss in k(cat), but 15- to 23-fold increases in K(m)(dUMP). Small hydrophobic substitutions for Pro197 were most active, and the most conservative mutant, P197A, had only a 5-fold lower k(cat)/K(m)(dUMP) than wild-type TS. Several Pro196 and Pro197 variants were temperature sensitive. The small effects of Pro196 or Pro197 mutations on enzyme kinetics suggest that the conformational restrictions encoded by the Pro-Pro sequence are largely maintained when either member of the pair is mutated.     

Use of amber suppressors to investigate the thermostability of Bacillus licheniformis alpha-amylase. Amino acid replacements at 6 histidine residues reveal a critical position at His-133

A set of 12 Escherichia coli suppressor tRNAs, inserting different amino acids in response to an amber codon, has been used to create rapidly numerous protein variants of a thermostable amylase; by site-directed mutagenesis, amber mutations were first introduced into Bacillus licheniformis alpha-amylase gene at position His35, His133, His247, His293, His406, or His450; genes carrying one or two amber mutations were then expressed in the different suppressor strains, generating over 100 amylase variants with predicted amino acid changes that could be tested for thermostability. Within the detection limits of the assays, amino acid replacements at five histidine positions had no significant effect. In contrast, suppressed variants substituted at residue His133 clearly exhibited modified thermostability and could be either less stable or more stable than the wild-type amylase, depending on the amino acid inserted at this position; comparison of the variants indicates that the hydrophobicity of the substituting residue is an important but not a determinant factor of stabilization. The effect of the most stabilizing and destabilizing amino acid substitutions, His133 to Tyr and to Pro, respectively, were confirmed by introducing the corresponding missense mutations into the gene sequence. The advantages and limits of informational suppression in protein stability studies are discussed as well as structural features involved in the thermostability of B. licheniformis alpha-amylase.     

Cold adaptation of a psychrophilic chitinase: a mutagenesis study

The gene encoding chitinase ArChiB from the Antarctic Arthrobacter sp. TAD20 has been expressed in Escherichia coli and the recombinant enzyme purified to homogeneity. In an effort to engineer cold-adapted biocatalysts through rational redesign to operate at elevated temperatures, we performed several mutations aiming to increase the rigidity of the molecular edifice of the selected psychrophilic chitinase. The mutations were designed on the basis of a homology-based three-dimensional model of the enzyme, and included an attempt to introduce a salt bridge (mutant N198K) and replacements of selected Gly residues by either Pro (mutants G93P, G254P) or Gln (G406Q). Mutant N198K resulted in a more stable protein (DeltaTm = 0.6 degrees C). Mutant G93P exhibited a DeltaTm of 1.2 degrees C, while mutants G254P and G406Q exhibited decreased stability. We conclude that the effect of mutating Gly residues on enzyme stability is rather complex and can only be understood in the context of the structural environment. Kinetic and spectroscopic analysis of these enzyme variants revealed that the kinetic parameters kcat and Km have been significantly modified.     

Identification of functionally relevant histidine residues in the apoptotic nuclease CAD

The caspase-activated DNase CAD (DFF40/CPAN) degrades chromosomal DNA during apoptosis. Chemical modification with DEPC inactivates the enzyme, suggesting that histidine residues play a decisive role in the catalytic mechanism of this nuclease. Sequence alignment of murine CAD with four homologous apoptotic nucleases reveals four completely (His242, His263, His304 and His308) and two partially (His127 and His313) conserved histidine residues in the catalytic domain of the enzyme. We have changed these residues to asparagine and characterised the variant enzymes with respect to their DNA cleavage activity, structural integrity and oligomeric state. All variants show a decrease in activity compared to the wild-type nuclease as measured by a plasmid DNA cleavage assay. H242N, H263N and H313N exhibit DNA cleavage activities below 5% and H308N displays a drastically altered DNA cleavage pattern compared to wild-type CAD. Whereas all variants but one have the same secondary structure composition and oligomeric state, H242N does not, suggesting that His242 has an important structural role. On the basis of these results, possible roles for His127, His263, His304, His308 and His313 in DNA binding and cleavage are discussed for murine CAD.     

Identification of structural elements important for matrix metalloproteinase type V collagenolytic activity as revealed by chimeric enzymes. Role of fibronectin-like domain and active site of gelatinase B

Digestion of type V collagen by the gelatinases is an important step in tumor cell metastasis because this collagen maintains the integrity of the extracellular matrix that must be breached during this pathological process. However, the structural elements that provide the gelatinases with this unique proteolytic activity among matrix metalloproteinases had not been thoroughly defined. To identify these elements, we examined the substrate specificity of chimeric enzymes containing domains of gelatinase B and fibroblast collagenase. We have found that the addition of the fibronectin-like domain of gelatinase B to fibroblast collagenase is sufficient to endow the enzyme with the ability to cleave type V collagen. In addition, the substitution of the catalytic zinc-binding active site region of fibroblast collagenase with that of gelatinase B increased the catalytic efficiency of the enzyme 3- to 4-fold. This observation led to the identification of amino acid residues, Leu(397), Ala(406), Asp(410), and Pro(415), in this region of gelatinase B that are important for its efficient catalysis as determined by substituting these amino acids with the corresponding residues from fibroblast collagenase. Leu(397) and Ala(406) are important for the general proteolytic activity of the enzyme, whereas Asp(410) and Pro(415) specifically enhance its ability to cleave type V collagen and gelatin, respectively. These data provide fundamental information about the structural elements that distinguish the gelatinases from other matrix metalloproteinases in terms of substrate specificity and catalytic efficiency.     

Tyrosine residues near the FAD binding site are critical for FAD binding and for the maintenance of the stable and active conformation of rat monoamine oxidase A.

Monoamine oxidase is a flavin-containing enzyme located at the mitochondrial outer membrane that catalyzes the oxidative deamination of amines. To investigate the role of tyrosine residues near the FAD-binding site, Cys-406, of monoamine oxidase A, the tyrosine residues at posiyions 402, 407, and 410 were indurdually replaced with alanine or phenylalanine and the effects of the mutations on catalytic activity, FAD binding, and enzyme structure were examined. Half or fewer of the mutant proteins incorporated FAD. The mutation of Tyr-407 to alanine led to an almost completely loss of catalytic activity for serotonin, PEA, tyramine, and tryptamine. A substantial decrease in the catalytic activity was also observed with the enzymes mutated at Tyr-402 and Tyr-410 to alanine, although the effect of the latter mutation was much less. All these mutants were sensitive to trypsin treatment of the purified enzyme, while the wild type enzyme was resistant to treatment. On the other hand, substitution of Tyr-402 or Tyr-407 with phenylalanine had little effect on these properties. Taken together, we conclude that tyrosine residues near Cys-406 may be form a pocket to facilitates FAD incorporation, the catalytic center, and a stable conformation, probably through interactions among the aromatic rings of the tyrosine residues and FAD.     

Dissecting structural and electrostatic interactions of charged groups in alpha-sarcin. An NMR study of some mutants involving the catalytic residues

The cytotoxic ribonuclease alpha-sarcin is the best characterized member of the ribotoxin family. Ribotoxins share a common structural core, catalytic residues, and active site topology with members of the broader family of nontoxic microbial extracellular RNases. They are, however, much more specific in their biological action. To shed light on the highly specific alpha-sarcin activity, we have evaluated the structural and electrostatic interactions of its charged groups, by combining the structural and pK(a) characterization by NMR of several variants with theoretical calculations based on the Tanford-Kirkwood and Poisson-Boltzmann models. The NMR data reveal that the global conformation of wild-type alpha-sarcin is preserved in the H50Q, E96Q, H137Q, and H50/137Q variants, and that His137 is involved in an H-bond that is crucial in maintaining the active site structure and in reinforcing the stability of the enzyme. The loss of this H-bond in the H137Q and H50/137Q variants modifies the local structure of the active site. The pK(a) values of active site groups H50, E96, and H137 in the four variants have been determined by two-dimensional NMR. The catalytic dyad of E96 and H137 is not sensitive to charge replacements, since their pK(a) values vary less than +/-0.3 pH unit with respect to those of the wild type. On the contrary, the pK(a) of His50 undergoes drastic changes when compared to its value in the intact protein. These amount to an increase of 0.5 pH unit or a decrease of 1.1 pH units depending on whether a positive or negative charge is substituted at the active site. The main determinants of the pK(a) values of most of the charged groups in alpha-sarcin have been established by considering the NMR results in conjunction with those derived from theoretical pK(a) calculations. With regard to the active site residues, the H50 pK(a) is chiefly influenced by electrostatic interactions with E96 and H137, whereas the effect of the low dielectric constant and the interaction with R121 appear to be the main determinants of the altered pK(a) value of E96 and H137. Charge-charge interactions and an increased level of burial perturb the pK(a) values of the active site residues of alpha-sarcin, which can account for its reduced ribonucleolytic activity and its high specificity.     

Kinetic and structural characterization of urease active site variants

Klebsiella aerogenes urease uses a dinuclear nickel active site to catalyze urea hydrolysis at >10(14)-fold the spontaneous rate. To better define the enzyme mechanism, we examined the kinetics and structures for a suite of site-directed variants involving four residues at the active site: His320, His219, Asp221, and Arg336. Compared to wild-type urease, the H320A, H320N, and H320Q variants exhibit similar approximately 10(-)(5)-fold deficiencies in rates, modest K(m) changes, and disorders in the peptide flap covering their active sites. The pH profiles for these mutant enzymes are anomalous with optima near 6 and shoulders that extend to pH 9. H219A urease exhibits 10(3)-fold increased K(m) over that of native enzyme, whereas the increase is less marked ( approximately 10(2)-fold) in the H219N and H219Q variants that retain hydrogen bonding capability. Structures for these variants show clearly resolved active site water molecules covered by well-ordered peptide flaps. Whereas the D221N variant is only moderately affected compared to wild-type enzyme, D221A urease possesses low activity ( approximately 10(-)(3) that of native enzyme), a small increase in K(m), and a pH 5 optimum. The crystal structure for D221A urease is reminiscent of the His320 variants. The R336Q enzyme has a approximately 10(-)(4)-fold decreased catalytic rate with near-normal pH dependence and an unaffected K(m). Phenylglyoxal inactivates the R336Q variant at over half the rate observed for native enzyme, demonstrating that modification of non-active-site arginines can eliminate activity, perhaps by affecting the peptide flap. Our data favor a mechanism in which His219 helps to polarize the substrate carbonyl group, a metal-bound terminal hydroxide or bridging oxo-dianion attacks urea to form a tetrahedral intermediate, and protonation occurs via the general acid His320 with Asp221 and Arg336 orienting and influencing the acidity of this residue. Furthermore, we conclude that the simple bell-shaped pH dependence of k(cat) and k(cat)/K(m) for the native enzyme masks a more complex underlying pH dependence involving at least four pK(a)s.     

Catalytic role of the active site histidine of porcine pancreatic phospholipase A2 probed by the variants H48Q, H48N and H48K.

The catalytic contribution of His48 in the active site of porcine pancreatic phospholipase A2 was examined using site-directed mutagenesis. Replacement of His48 by lysine (H48K) gives rise to a protein having a distorted lipid binding pocket. Activity of this variant drops below the detection limit which is 10(7)-fold lower than that of the wild-type enzyme. On the other hand, the presence of glutamine (H48Q) or asparagine (H48N) at this position does not affect the structural integrity of the enzyme as can be derived from the preserved lipid binding properties of these variants. However, the substitutions H48Q and H48N strongly reduce the turnover number, i.e. by a factor of 10(5). Residual activity is totally lost after addition of a competitive inhibitor. We conclude that proper lipid binding on its own accelerates ester bond hydrolysis by a factor of 10(2). With the selected variants, we were also able to dissect the contribution of the hydrogen bond between Asp99 and His48 on conformational stability, being 5.2 kJ/mol. Another hydrogen bond with His48 is formed when the competitive inhibitor (R)-2-dodecanoylamino-hexanol-1-phosphoglycol interacts with the enzyme. Its contribution to binding of the inhibitor in the presence of an interface was found to be 5.7 kJ/mol.     

Site-directed mutagenesis of selected residues at the active site of aryl-alcohol oxidase, an H2O2-producing ligninolytic enzyme

Aryl-alcohol oxidase provides H(2)O(2) for lignin biodegradation, a key process for carbon recycling in land ecosystems that is also of great biotechnological interest. However, little is known of the structural determinants of the catalytic activity of this fungal flavoenzyme, which oxidizes a variety of polyunsaturated alcohols. Different alcohol substrates were docked on the aryl-alcohol oxidase molecular structure, and six amino acid residues surrounding the putative substrate-binding site were chosen for site-directed mutagenesis modification. Several Pleurotus eryngii aryl-alcohol oxidase variants were purified to homogeneity after heterologous expression in Emericella nidulans, and characterized in terms of their steady-state kinetic properties. Two histidine residues (His502 and His546) are strictly required for aryl-alcohol oxidase catalysis, as shown by the lack of activity of different variants. This fact, together with their location near the isoalloxazine ring of FAD, suggested a contribution to catalysis by alcohol activation, enabling its oxidation by flavin-adenine dinucleotide (FAD). The presence of two aromatic residues (at positions 92 and 501) is also required, as shown by the conserved activity of the Y92F and F501Y enzyme variants and the strongly impaired activity of Y92A and F501A. By contrast, a third aromatic residue (Tyr78) does not seem to be involved in catalysis. The kinetic and spectral properties of the Phe501 variants suggested that this residue could affect the FAD environment, modulating the catalytic rate of the enzyme. Finally, L315 affects the enzyme k(cat), although it is not located in the near vicinity of the cofactor. The present study provides the first evidence for the role of aryl-alcohol oxidase active site residues.     

Molecular dynamic simulations of nanomechanic chaperone peptide and effects of in silico His mutations on nanostructured function

The nanoscale peptide YSGVCHTDLHAWHGDWPLPVK exhibits molecular chaperone activity and prevents protein aggregation under chemical and/or thermal stress. Here, His mutations of this peptide and their impact on chaperone activity were evaluated using theoretical techniques. Molecular dynamic (MD) simulations with simulated annealing (SA) of different mutant nanopeptides were employed to determine the contribution of the scaffolding His residues (H45, H49, H52), when mutated to Pro, on chaperone action in vitro. The in silico mutations of His residues to Pro (H45P, H49P, H52P) revealed loss of secondary ordered strand structure. However, a small part of the strand conformation was formed in the middle region of the native chaperone peptide. The His‐to‐Pro mutations resulted in decreased gyration radius (R_g) values and surface accessibility of the mutant peptides under the simulation times. The invariant dihedral angle (ϕ) values and the disrupting effects of the Pro residues indicated the coil conformation of mutant peptides. The failure of the chaperone‐like action in the Pro mutant peptides was consistent with their decreased effective accessible surfaces. The high variation of Φ value for His residues in native chaperone peptide leads to high flexibility, such as a minichaperone acting as a nanomachine at the molecular level. Our findings demonstrate that the peptide strand conformation motif with high flexibility at nanoscale is critical for chaperone activity. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Identification of the transmembrane metal binding site in Cu+-transporting PIB-type ATPases

P(IB)-type ATPases have an essential role maintaining copper homeostasis. Metal transport by these membrane proteins requires the presence of a transmembrane metal occlusion/binding site. Previous studies showed that Cys residues in the H6 transmembrane segment are required for metal transport. In this study, the participation in metal binding of conserved residues located in transmembrane segments H7 and H8 was tested using CopA, a model Cu(+)-ATPase from Archaeoglobus fulgidus. Four invariant amino acids in the central portion of H7 (Tyr(682) and Asn(683)) and H8 (Met(711) and Ser(715)) were identified as required for Cu(+) binding. Replacement of these residues abolished enzyme activity. These proteins did not undergo Cu(+)-dependent phosphorylation by ATP but were phosphorylated by P(i) in the absence of Cu(+). Moreover, the presence of Cu(+) could not prevent the enzyme phosphorylation by P(i). Other conserved residues in the H7-H8 region were not required for metal binding. Mutation of two invariant Pro residues had little effect on enzyme function. Replacement of residues located close to the cytoplasmic end of H7-H8 led to inactive enzymes. However, these were able to interact with Cu(+) and undergo phosphorylation. This suggests that the integrity of this region is necessary for conformational transitions but not for ligand binding. These data support the presence of a unique transmembrane Cu(+) binding/translocation site constituted by Tyr-Asn in H7, Met and Ser in H8, and two Cys in H6 of Cu(+)-ATPases. The likely Cu(+) coordination during transport appears distinct from that observed in Cu(+) chaperone proteins or catalytic/redox metal binding sites.     

Enhancing thermostability and the structural characterization of Microbacterium saccharophilum K-1 β-fructofuranosidase

A β-fructofuranosidase from Microbacterium saccharophilum K-1 (formerly known as Arthrobacter sp. K-1) is useful for producing the sweetener lactosucrose (4^G-β-d-galactosylsucrose). Thermostability of the β-fructofuranosidase was enhanced by random mutagenesis and saturation mutagenesis. Clones with enhanced thermostability included mutations at residues Thr47, Ser200, Phe447, Phe470, and Pro500. In the highest stability mutant, T47S/S200T/F447P/F470Y/P500S, the half-life at 60 °C was 182 min, 16.5-fold longer than the wild-type enzyme. A comparison of the crystal structures of the full-length wild-type enzyme and three mutants showed that various mechanisms appear to be involved in thermostability enhancement. In particular, the replacement of Phe447 with Val or Pro induced a conformational change in an adjacent residue His477, which results in the formation of a new hydrogen bond in the enzyme. Although the thermostabilization mechanisms of the five residue mutations were explicable on the basis of the crystal structures, it appears to be difficult to predict which amino acid residues should be modified to obtain thermostabilized enzymes.     

A phosphate-binding subsite in bovine pancreatic ribonuclease A can be converted into a very efficient catalytic site

A general acid-base catalytic mechanism is responsible for the cleavage of the phosphodiester bonds of the RNA by ribonuclease A (RNase A). The main active site is formed by the amino acid residues His12, His119, and Lys41, and the process follows an endonucleolytic pattern that depends on the existence of a noncatalytic phosphate-binding subsite adjacent, on the 3'-side, to the active site; in this region the phosphate group of the substrate establishes electrostatic interactions through the side chains of Lys7 and Arg10. We have obtained, by means of site-directed mutagenesis, RNase A variants with His residues both at positions 7 and 10. These mutations have been introduced with the aim of transforming a noncatalytic binding subsite into a putative new catalytic active site. The RNase activity of these variants was determined by the zymogram technique and steady-state kinetic parameters were obtained by spectrophotometric methods. The variants showed a catalytic efficiency in the same order of magnitude as the wild-type enzyme. However, we have demonstrated in these variants important effects on the substrate's cleavage pattern. The quadruple mutant K7H/R10H/H12K/H119Q shows a clear increase of the exonucleolytic activity; in this case the original native active site has been suppressed, and, as consequence, its activity can only be associated to the new active site. In addition, the mutant K7H/R10H, with two putative active sites, also shows an increase in the exonucleolytic preference with respect to the wild type, a fact that may be correlated with the contribution of the new active site.     

Mutational analysis of Cvab, an ABC transporter involved in the secretion of active colicin V

CvaB is the central membrane transporter of the colicin V secretion system that belongs to an ATP-binding cassette superfamily. Previous data showed that the N-terminal and C-terminal domains of CvaB are essential for the function of CvaB. N-terminal domain of CvaB possesses Ca(2+)-dependent cysteine proteolytic activity, and two critical residues, Cys32 and His105, have been identified. In this study, we also identify Asp121 as being the third residue of the putative catalytic triad within the active site of the enzyme. The Asp121 mutants lose both their colicin V secretion activity and N-terminal proteolytic activity. The adjacent residue Pro122 also appears to play a critical role in the colicin V secretion. However, the reversal of the two residues D121P - P122D results in loss of activity. Based on molecular modeling and protein sequence alignment, several residues adjacent to the critical residues, Cys32 and His105, were also examined and characterized. Site-directed mutagenesis of Trp101, Asp102, Val108, Leu76, Gly77, and Gln26 indicate that the neighboring residues around the catalytic triad affect colicin V secretion. Several mutated CvaB proteins with defective secretion were also tested, including Asp121 and Pro122, and were found to be structurally stable. These results indicate that the residues surrounding the identified catalytic triad are functionally involved in the secretion of biologically active colicin V.     

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn(2+) binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.     

Role of proline residues in conferring thermostability on aqualysin I

To understand the molecular basis of the thermostability of a thermophilic serine protease aqualysin I from Thermus aquaticus YT-1, we introduced mutations at Pro5, Pro7, Pro240 and Pro268, which are located on the surface loops of aqualysin I, by changing these amino acid residues into those found at the corresponding locations in VPR, a psychrophilic serine protease from Vibrio sp. PA-44. All mutants were expressed stably and exhibited essentially the same specific activity as wild-type aqualysin I at 40 degrees C. The P240N mutant protein had similar thermostability to wild-type aqualysin I, but P5N and P268T showed lower thermostability, with a half-life at 90 degrees C of 15 and 30 min, respectively, as compared to 45 min for the wild-type enzyme. The thermostability of P7I was decreased even more markedly, and the mutant protein was rapidly inactivated at 80 degrees C and even at 70 degrees C, with half-lives of 10 and 60 min, respectively. Differential scanning calorimetry analysis showed that the transition temperatures of wild-type enzyme, P5N, P7I, P240N and P268T were 93.99 degrees C, 83.45 degrees C, 75.66 degrees C, 91.78 degrees C and 86.49 degrees C, respectively. These results underscore the importance of the proline residues in the N- and C-terminal regions of aqualysin I in maintaining the integrity of the overall protein structure at elevated temperatures.     

Proline effect on the thermostability and slow unfolding of a hyperthermophilic protein

Ribonuclease HII from hyperthermophile Thermococcus kodakaraensis (Tk-RNase HII) is a robust monomeric protein under kinetic control, which possesses some proline residues at the N-terminal of alpha-helices. Proline residue at the N-terminal of an alpha-helix is thought to stabilize a protein. In this work, the thermostability and folding kinetics of Tk-RNase HII were measured for mutant proteins in which a proline residue is introduced (Xaa to Pro) or removed (Pro to Ala) at the N-terminal of alpha-helices. In the folding experiments, the mutant proteins examined exhibit little influence on the remarkably slow unfolding of Tk-RNase HII. In contrast, E111P and K199P exhibit some thermostabilization, whereas P46A, P70A and P174A have some thermodestabilization. E111P/K199P and P46A/P70A double mutations cause cumulative changes in stability. We conclude that the proline effect on protein thermostability is observed in a hyperthermophilic protein, but each proline residue at the N-terminal of an alpha-helix slightly contributes to the thermostability. The present results also mean that even a natural hyperthermophilic protein can acquire improved thermostability.     

Stringency of the 2-His–1-Asp Active-Site Motif in Prolyl 4-Hydroxylase

The non-heme iron(II) dioxygenase family of enzymes contain a common 2-His–1-carboxylate iron-binding motif. These enzymes catalyze a wide variety of oxidative reactions, such as the hydroxylation of aliphatic C–H bonds. Prolyl 4-hydroxylase (P4H) is an α-ketoglutarate-dependent iron(II) dioxygenase that catalyzes the post-translational hydroxylation of proline residues in protocollagen strands, stabilizing the ensuing triple helix. Human P4H residues His412, Asp414, and His483 have been identified as an iron-coordinating 2-His–1-carboxylate motif. Enzymes that catalyze oxidative halogenation do so by a mechanism similar to that of P4H. These halogenases retain the active-site histidine residues, but the carboxylate ligand is replaced with a halide ion. We replaced Asp414 of P4H with alanine (to mimic the active site of a halogenase) and with glycine. These substitutions do not, however, convert P4H into a halogenase. Moreover, the hydroxylase activity of D414A P4H cannot be rescued with small molecules. In addition, rearranging the two His and one Asp residues in the active site eliminates hydroxylase activity. Our results demonstrate a high stringency for the iron-binding residues in the P4H active site. We conclude that P4H, which catalyzes an especially demanding chemical transformation, is recalcitrant to change.