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.     

Kinetic stabilization of Bacillus licheniformis alpha-amylase through introduction of hydrophobic residues at the surface

It is generally assumed that in proteins hydrophobic residues are not favorable at solvent-exposed sites, and that amino acid substitutions on the surface have little effect on protein thermostability. Contrary to these assumptions, we have identified hyperthermostable variants of Bacillus licheniformis alpha-amylase (BLA) that result from the incorporation of hydrophobic residues at the surface. Under highly destabilizing conditions, a variant combining five stabilizing mutations unfolds 32 times more slowly and at a temperature 13 degrees C higher than the wild-type. Crystal structure analysis at 1.7 A resolution suggests that stabilization is achieved through (a) extension of the concept of increased hydrophobic packing, usually applied to cavities, to surface indentations, (b) introduction of favorable aromatic-aromatic interactions on the surface, (c) specific stabilization of intrinsic metal binding sites, and (d) stabilization of a beta-sheet by introducing a residue with high beta-sheet forming propensity. All mutated residues are involved in forming complex, cooperative interaction networks that extend from the interior of the protein to its surface and which may therefore constitute "weak points" where BLA unfolding is initiated. This might explain the unexpectedly large effect induced by some of the substitutions on the kinetic stability of BLA. Our study shows that substantial protein stabilization can be achieved by stabilizing surface positions that participate in underlying cooperatively formed substructures. At such positions, even the apparently thermodynamically unfavorable introduction of hydrophobic residues should be explored.     

Identification of critical amino acid residues for chloride binding of Bacillus licheniformis trehalose-6-phosphate hydrolase

Based on sequence alignment of selected Cl^? dependent and independent glycoside hydrolase family 13 enzymes, two invariant residues (Arg201 and Asn347) and one tyrosine (Tyr365) that might be responsible for the binding of Bacillus licheniformis trehalose-6-phosphate hydrolase (BlTreA) to chloride ion were identified. The role of these three residues was further explored by mutational and biophysical analyses. The mutant enzymes (R201Q/E/K, N327Q/D/K, and Y365A/R) and BlTreA were individually overexpressed in Escherichia coli M15 host cells and purified by one-step nickel affinity chromatography on Ni-NTA resin. The purified BlTreA and Y365A had a specific activity of 236.9 and 47.6 U/mg protein, respectively. The remaining enzymes lost their hydrolase activity completely even in the presence of high salt. With the exception of Y365A, all mutant enzymes did not have the ability to bind fluoride, chloride and nitrate anions. Structural analyses showed that the circular dichroism spectra of the mutant proteins were consistent with those of BlTreA. However, wild-type and mutant enzymes displayed a slight difference in the profiles of intrinsic tryptophan fluorescence. Collectively, these results clearly indicate that Arg201 and Agr327 residues might play an essential role in chloride binding of BlTreA.     

Amino acid substitution in the lactose carrier protein with the use of amber suppressors.

Five lacY mutants with amber stop codons at known positions were each placed into 12 different suppressor strains. The 60 amino acid substitutions obtained in this manner were tested for growth on lactose-minimal medium plates and for transport of lactose, melibiose, and thiomethylgalactoside. Most of the amino acid substitutions in the regions of the putative loops (between transmembrane alpha helices) resulted in a reasonable growth rate on lactose with moderate-to-good transport activity. In one strain (glycine substituted for Trp-10), abnormal sugar recognition was found. The substitution of proline for Trp-33 (in the region of the first alpha helix) showed no activity, while four additional substitutions (lysine, leucine, cysteine, and glutamic acid) showed low activity. Altered sugar specificity was observed when Trp-33 was replaced by serine, glutamine, tyrosine, alanine, histidine, or phenylalanine. It is concluded that Trp-33 may be involved directly or indirectly in sugar recognition.     

Identification of essential histidine residues in a recombinant alpha-amylase of thermophilic and alkaliphilic Bacillus sp. strain TS-23

To understand the structure-function relationships of a truncated Bacillus sp. strain TS-23 -amylase, each of His-137, His-191, His-239, His-269, His-305, His-323, His-361, His-436, and His-475 was replaced with leucine. The molecular masses of the purified wild-type and mutant enzymes were approximately 54 kDa. The specific activity of His323Leu and His436Leu was decreased by more than 52%, while His239Leu, His305Leu, and His475Leu showed activity similar to that of the wild-type enzyme. As compared with the wild-type enzyme, His323Leu and His436Leu exhibited a 62% decrease in the value of k_cat/K_m. Alterations in His-191, His-239, His-305, and His-475 did not cause a significant change in the K_m or k_cat values. At 70°C, a decreased half-life was observed in His436Leu. These results indicate that His-137, His-269, and His-361 of Bacillus sp. strain TS-23 -amylase are important for proper catalytic activity and that His-436 may contribute to the thermostability of the enzyme.Communicated by K. Horikoshi     

Bivalent cations and amino-acid composition contribute to the thermostability of Bacillus licheniformis xylose isomerase.

Comparative analysis of genome sequence data from mesophilic and hyperthermophilic micro-organisms has revealed a strong bias against specific thermolabile amino-acid residues (i.e. N and Q) in hyperthermophilic proteins. The N + Q content of class II xylose isomerases (XIs) from mesophiles, moderate thermophiles, and hyperthermophiles was examined. It was found to correlate inversely with the growth temperature of the source organism in all cases examined, except for the previously uncharacterized XI from Bacillus licheniformis DSM13 (BLXI), which had an N + Q content comparable to that of homologs from much more thermophilic sources. To determine whether BLXI behaves as a thermostable enzyme, it was expressed in Escherichia coli, and the thermostability and activity properties of the recombinant enzyme were studied. Indeed, it was optimally active at 70-72 degrees C, which is significantly higher than the optimal growth temperature (37 degrees C) of B. licheniformis. The kinetic properties of BLXI, determined at 60 degrees C with glucose and xylose as substrates, were comparable to those of other class II XIs. The stability of BLXI was dependent on the metallic cation present in its two metal-binding sites. The enzyme thermostability increased in the order apoenzyme < Mg2+-enzyme < Co2+-enzyme approximately Mn2+-enzyme, with melting temperatures of 50.3 degrees C, 53.3 degrees C, 73.4 degrees C, and 73.6 degrees C. BLXI inactivation was first-order in all conditions examined. The energy of activation for irreversible inactivation was also strongly influenced by the metal present, ranging from 342 kJ x mol(-1) (apoenzyme) to 604 kJ x mol(-1) (Mg2+-enzyme) to 1166 kJ x mol(-1) (Co2+-enzyme). These results suggest that the first irreversible event in BLXI unfolding is the release of one or both of its metals from the active site. Although N + Q content was an indicator of thermostability for class II XIs, this pattern may not hold for other sets of homologous enzymes. In fact, the extremely thermostable alpha-amylase from B. licheniformis was found to have an average N + Q content compared with homologous enzymes from a variety of mesophilic and thermophilic sources. Thus, it would appear that protein thermostability is a function of more complex molecular determinants than amino-acid content alone.     

Amino acid residues adjacent to the catalytic cavity of tetramer l-asparaginase II contribute significantly to its catalytic efficiency and thermostability

l-Asparaginase (l-asparagine amidohydrolase, EC 3.5.1.1) catalyzes the hydrolysis of l-asparagine to l-aspartic acid and ammonia. It can be used to reduce the formation of acrylamide, which is carcinogenic to humans in foods, via removal of the precursor, asparagine, from the primary ingredients. However, low activity and poor thermostability of l-asparaginase restrict its application in food industry. In this study, we successfully improved thermostability and catalytic efficiency of l-asparaginase II (BsAII) from Bacillus subtilis B11-06 by site-directed mutagenesis. According to sequences alignment and homologous modeling, residues G107, T109 and S166 which were adjacent to the catalytic cavity were selected and substituted by Asp, Gln/Ser and Ala, respectively, to construct mutants G107D, T109Q, T109S and S166A. The BsAII mutant of G107D (G107D_ansz) displayed superior performance in thermal tolerance and higher activity than the wild-type enzyme (towards l-asparagine). Comparative analysis of hydrogen bond interactions, surface electrostatic potential and structure of substrate binding pocket between G107D_anszand BsAII indicated that the substitution of G107, which was adjacent to catalytic cavity with Asp, resulted in small conformational changes and surface electrostatic potential redistribution and contributed to the improved protein stability and catalytic efficiency.     

Use of the beta-lactamase inhibitor clavulanic acid in the isolation of auxotrophic mutants of Bacillus licheniformis.

The isolation of auxotrophic mutants of Bacillus licheniformis, a microbe containing constitutive beta-lactamase activity, was found to be facilitated by the addition of clavulanic acid and cefotaxime during enrichment.     

Amino acid replacements of the evolutionarily invariant tryptophan at position 64 in mutant forms of iso-1-cytochrome c from Saccharomyces cerevisiae

Tryptophan located at position 59 in vertebrate cytochromes c and at position 64 in yeast iso-1-cytochrome c is an evolutionarily invariant residue that is believed to be essential to the operation of the cytochrome c molecule. We show that this residue is replaced in at least partially functional iso-1-cytochromes c from cyc1 revertants of the yeast Saccharomyces cerevisiae. Tryptophan, tyrosine and leucine are found at position 64 in the revertants from the cyc1-84 mutant, confirming the genetic evidence (Sherman et al., 1974) that the mutant contains an UAG nonsense codon and establishing that the site of the mutation corresponds to the normal tryptophan 64. In a revertant from the cyc1.189 mutant, position 64 is occupied by a residue of phenylalanine. All three altered proteins are unstable, implying that tryptophan 64 has an essential and unique role for maintaining the normal structure of the cytochrome c molecule. In addition the iso-1-cytochrome c with leucine 64 and tyrosine 64 have greatly reduced biological activities, while iso-1-cytochrome c with the phenylalanine replacement has at least 20% of the wild-type activity or more. It remains uncertain whether the reduced specific activities are due to distorted tertiary structures or due to the specific lack of the tryptophan residue that may also have a direct functional role.     

Use of a new integrational vector to investigate compartment-specific expression of the Bacillus subtilis spoIIM gene.

The product of the spoIIM gene of Bacillus subtilis is required for complete septum migration and forespore engulfment during sporulation. To investigate whether expression of spoIIM is required in the forespore compartment of the sporangium, we have constructed a new integrational vector, pKSV7, which contains temperature-sensitive replication functions derived from pE194ts. The presence of the conditionally defective replication origin greatly stimulates plasmid excision when sporulation occurs at the permissive temperature. This facilitates the use of a genetic technique employed by Illing et al to distinguish genes whose expression must occur in the forespore from genes that may be expressed exclusively in the mother cell compartment. The results of the integration/excision experiments using pKSV7 support the conclusion that spoIIM must be expressed in the forespore. Biochemical analysis of forespore and mother cell fractions suggests that spoIIM is also expressed in the mother cell. The conditional integrational vector pKSV7 replicates at high copy number in E coli and allows the identification of inserts in the polylinker cluster by disruption of alpha-complementation and thus should be useful for other kinds of genetic manipulations in B subtilis.     

Role of teichuronic acid in Bacillus licheniformis: defective autolysis due to deficiency of teichuronic acid in a novobiocin-resistant mutant.

nov-12, a novobiocin-resistant mutant of Bacillus licheniformis ATCC 9945, grows as long chains of cells, a characteristic of autolytic-deficient (Lyt-) mutants. Isolated walls from nov-12 autolyzed at a rate equal to 5% of that displayed by wild-type walls, thus confirming the Lyt- phenotype. Protein-free nov-12 walls displayed marked resistance to, and also failure to bind, added autolysin solubilized from wild-type walls. Comparison of isolated cell walls revealed a deficiency in teichuronic acid in the mutant. Lesser differences were observed in walls of this strain, including a reduction in galactose, an increase in the proportion of peptidoglycan, and small quantitative differences in peptidoglycan composition though the proportions of protein and teichoic acid were similar in walls of both strains. Autolytic sensitivity was studied in walls in which protein, teichoic acid, and teichuronic acid were removed successively by selective extraction procedures. Autolysis of wild-type walls was unaffected by removal or protein or teichoic acid, but teichuronic acid removal rendered wild-type walls as insensitive to autolysis as mutant walls had been throughout. Therefore, in this mutant, deficiency in teichuronic acid alone leads to the Lyt- phenotype and, hence, activity and binding of autolysin(s) are dependent upon teichuronic acid but not teichoic acid. Also, the potential rate of autolysis of cell walls in this organism was correlated with the proportion of teichuronic acid in the wall. The possible significance of these findings with respect to control of autolysis and cell separation is discussed.     

The critical role of amino acid residue at position 117 of mouse UDP-glucuronosyltransfererase 1a6a and 1a6b in resveratrol glucuronidation

Mouse UDP-glucuronosyltransferase 1a6 (Ugt1a6) contains two functional copies of 1a6a and 1a6b that share high sequence homology (98%). Only 10 amino acids located around the substrate recognition region are different out of 531 total residues. Although Ugt1a6 plays important roles in conjugating phenolic compounds, the functional characteristics of these isozymes are unclear. We performed functional analyses of mouse Ugt1a6a and Ugt1a6b using two isomeric polyphenols (trans- and cis-resveratrol). The cDNAs of mouse Ugt1a6a and Ugt1a6b were cloned and constructed as recombinant proteins using a yeast expression system, and kinetic parameters were evaluated. The wild-type Ugt1a6a and Ugt1a6b proteins catalysed trans- and cis-resveratrol 3-O-glucuronidation. Although the K(m) value for trans-resveratrol was significantly lower for Ugt1a6a compared with Ugt1a6b, the K(m) values for cis-resveratrol were comparable for the isozymes. Despite high sequence homology, significant kinetic differences were observed between the isozymes. To identify the critical residues for resveratrol glucuronidation, we constructed 10 variants of Ugt1a6a (T81P, N96R, H98Q, L100V, S104P, N115S, I117L, V118T, V119L and D120E). The I117L variant had Ugt1a6b-like enzymatic properties of K(m) in trans-resveratrol, and V(max) and K(si) in cis-form, suggesting that the residues located at position 117 of Ugt1a6a and Ugt1a6b play an important role in resveratrol glucuronidation.     

Amino acid residues before the hydrophobic region which are critical for membrane translocation of the N-terminal domain of synaptotagmin II.

We examined the fine structure of the type I signal-anchor sequence of synaptotagmin II, which has a 60-residue N-terminal domain followed by a hydrophobic region (H-region), focusing on the hinge region between the N-terminal and the H-regions. It was found that the charged or highly polar residues support the translocation of the N-terminal domain through the endoplasmic reticulum membrane at specific positions in the hinge. The residue requirement correlated with the turn propensity scale for transmembranes. It is suggested that a certain conformation, likely helical hairpin, in the hinge is critical for N-terminal domain translocation.     

Characterization of a new cell-bound alpha-amylase in Bacillus subtilis 168 Marburg that is only immunologically related to the exocellular alpha-amylase.

Immunoblot analysis of Bacillus subtilis cell extracts with polyclonal antibodies, raised against purified exocellular alpha-amylase, revealed one protein species of 82,000 Da. This protein was found even in cells in which the amyE gene, encoding exocellular alpha-amylase, was disrupted. Isolated from the membrane fraction, the 82,000-M(r) protein displayed an alpha-amylase activity in vitro.     

Engineering the substrate specificity of Escherichia coli asparaginase. II. Selective reduction of glutaminase activity by amino acid replacements at position 248

The use of Escherichia coli asparaginase II as a drug for the treatment of acute lymphoblastic leukemia is complicated by the significant glutaminase side activity of the enzyme. To develop enzyme forms with reduced glutaminase activity, a number of variants with amino acid replacements in the vicinity of the substrate binding site were constructed and assayed for their kinetic and stability properties. We found that replacements of Asp248 affected glutamine turnover much more strongly than asparagine hydrolysis. In the wild-type enzyme, N248 modulates substrate binding to a neighboring subunit by hydrogen bonding to side chains that directly interact with the substrate. In variant N248A, the loss of transition state stabilization caused by the mutation was 15 kJ mol(-1) for L-glutamine compared to 4 kJ mol(-1) for L-aspartic beta-hydroxamate and 7 kJ mol(-1) for L-asparagine. Smaller differences were seen with other N248 variants. Modeling studies suggested that the selective reduction of glutaminase activity is the result of small conformational changes that affect active-site residues and catalytically relevant water molecules.     

Identification of the amino acid residues critical for specific binding of the bacteriolytic enzyme of gamma-phage, PlyG, to Bacillus anthracis

Bacillus anthracis causes anthrax, a lethal disease affecting humans, which has attracted attention due to its bioterrorism potential. gamma-Phage specifically infects B. anthracis, and is used for its detection. gamma-Phage lysin, PlyG, specifically lyses B. anthracis. Mutational analysis of PlyGB (PlyG binding domain; residues 156-233) indicated that positions 190-199 are necessary for binding to B. anthracis. This region is the central part of PlyGB and is predicted to form a beta-sheet. The amino acid residues of this region are also conserved in other lysins specific for B. anthracis. Alanine substitution at position 190 or 199 within this region resulted in significantly reduced binding, suggesting that L190 and Q199 play key roles in binding of PlyGB to B. anthracis. Our observations provide new insight into the mechanism of specific binding of lysin to B. anthracis, and may be useful in establishing new methods for detection of B. anthracis.     

Identification of essential residues in 2',3'-cyclic nucleotide 3'-phosphodiesterase. Chemical modification and site-directed mutagenesis to investigate the role of cysteine and histidine residues in enzymatic activity

2',3'-Cyclic nucleotide 3'-phosphodiesterase (CNP; EC ) catalyzes in vitro hydrolysis of 3'-phosphodiester bonds in 2',3'-cyclic nucleotides to produce 2'-nucleotides exclusively. N-terminal deletion mapping of the C-terminal two-thirds of recombinant rat CNP1 identified a region that possesses the catalytic domain, with further truncations abolishing activity. Proteolysis and kinetic analysis indicated that this domain forms a compact globular structure and contains all of the catalytically essential features. Subsequently, this catalytic fragment of CNP1 (CNP-CF) was used for chemical modification studies to identify amino acid residues essential for activity. 5,5'-Dithiobis-(2-nitrobenzoic acid) modification studies and kinetic analysis of cysteine CNP-CF mutants revealed the nonessential role of cysteines for enzymatic activity. On the other hand, modification studies with diethyl pyrocarbonate indicated that two histidines are essential for CNPase activity. Consequently, the only two conserved histidines, His-230 and His-309, were mutated to phenylalanine and leucine. All four histidine mutants had k(cat) values 1000-fold lower than wild-type CNP-CF, but K(m) values were similar. Circular dichroism studies demonstrated that the low catalytic activities of the histidine mutants were not due to gross changes in secondary structure. Taken together, these results demonstrate that both histidines assume critical roles for catalysis.