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The effects of point mutations of the conserved Asp443, Glu478, Asn494, and Asp498 residues in the RNase H domain of human immunodeficiency virus type I (HIV-1) reverse transcriptase (RT) have been analyzed. The mutants fell into two classes: (i) functional RT, but not detectable ribonuclease H activity, and (ii) uncharacterizable phenotype due to protein instability in the context of the RT/protease Escherichia coli co-expression system (Mizrahi, V., Lazarus, G. M., Miles, L. M., Meyers, C. A., and Debouck, C. (1989) Arch. Biochem. Biophys. 273, 347-358). The only mutation in the former class was D443A, whereas those in the latter included D443E, E478D, E478Q, D498E, D443A/D498N, D443E/D498N, D443Q/D498N, N494A, N494D, and N494Q. The results were interpreted in terms of the x-ray crystal structure of the HIV-1 RNase H domain (Davies, J. F., II, Hostomaska, Z., Hostomsky, Z., Jordan, S. R., and Matthews, D. A. (1991) Science 252, 88-95) and a general acid-general base hydrolysis mechanism (Katayanagi, K., Okumura, M., and Morikawa, K. (1993) Proteins Struct. Funct. Genet. 17, 337-346). The data suggested that structural perturbations within the RNase H domain interfered with maturation of the pol precursor by HIV-1 protease. Analysis of selected D443/D498 double mutants suggested that the destabilization caused by the D498N mutation could be suppressed by the formation of a new hydrogen bond between Asn498 and Asn443.

Recently, several missense mutations in the Cu/Zn superoxide dismutase gene (SOD1) have been reported as a putative cause of chromosome-21q-linked familial amyotrophic lateral sclerosis (FALS). We have discovered a novel missense mutation (substitution of Thr for Ala4) in exon 1 (GCC to ACC) in two FALS patients from one Japanese FALS family. No mutations were found in 17 cases of sporadic ALS. The enzyme activity of recombinant fusion protein containing the Cu/Zn superoxide dismutase (SOD) with the Ala4-to-Thr mutation was significantly reduced in E. coli. On the other hand, in the expression system in insect cells using Baculovirus, the mutant SOD expressed an enzyme activity as high as wild-type SOD. These results suggest that the stability of SOD with the Ala4-to-Thr mutation is disrupted especially in the fusion protein. Autopsy was carried out on one of the two patients, and the pathological findings were typical of FALS with posterior column involvement. These results raise the possibility that mutation of the SOD1 is responsible for FALS with broader pathological involvement.

The 3-D structures of two chimeric enzymes (4M6T and 2T2M6T) between the Bacillus subtilis and Thermus thermophilus 3-isopropylmalate dehydrogenases were analysed by X-ray diffraction in order to investigate their different thermostabilities. The structure of 2T2M6T was determined by the difference Fourier method and that of 4M6T by rigid body refinement, as based on the structure of the T. thermophilus enzyme. These structures were refined stereochemically to an R-factor of 0.193 at 2.5 A resolution for 4M6T and to an R-factor of 0.195 at 2.2 A resolution for 2T2M6T. The 3-D structures of 4M6T and 2T2M6T were very close to the structure of the T. thermophilus enzyme, conspicuous differences being at the molecular surface. In particular, 2T2M6T having a larger reduction in thermostability was more closely related to the T. thermophilus enzyme. However, their correlations between C alpha-atom displacements and the root squares of the temperature factors were significantly different from each other.

Bacillus stearothermophilus esterase contains two free cysteine residues at positions of 45 and 115, which react with sulfhydryl reagents resulting in a significant decrease in the enzymatic activity. To understand the role of the cysteine residues in catalytic regions of the esterase, the residues were replaced with serine or alanine by site-directed mutagenesis to construct four single-mutated enzymes (C45A, C45S, C115A, C115S) and two double-mutated ones (C45/115A and C45/115S). Wild-type and mutant enzymes were produced in Escherichia coli cells and purified to homogeneity to examine their chemical and kinetic properties. These mutant enzymes had esterase activity, which suggested that none of the cysteines were required for its activity. Moreover, replacement of both two-cysteine residues made the enzyme insensitive to p-chloromercuribenzoic acid and extensively stabilized it at high temperatures of around 70 degrees C. These results demonstrate that replacement of free cysteine residues by site-directed mutagenesis can improve the thermostability of thermophilic enzymes.

An M13 bacteriophage-based in vivo screening system has been developed to identify T4 lysozyme mutants of enhanced thermal stability. This system takes advantage of easy mutagenesis in an M13 host, the production of functional T4 lysozyme during M13 growth, and the ability to detect lysozyme activity on agar plates. Of several mutagenesis procedures that were tested, the most efficient was based on misincorporation by avian myeloma virus reverse transcriptase. This one-step mutagenesis and screening system has been used to find 18 random single-site mutant lysozymes, of which 11 were heat resistant. Each of these had a melting temperature within 0.8-1.4 degrees C of wild type, suggesting that the screening system is quite sensitive.

The structures of three mutants of bacteriophage T4 lysozyme selected using a screen designed to identify thermostable variants are described. Each of the mutants has a substitution involving threonine. Two of the variants, Thr 26-->Ser (T26S) and Thr 151-->Ser (T151S), have increased reversible melting temperatures with respect to the wild-type protein. The third, Ala 93-->Thr (A93T), has essentially the same stability as wild type. Thr 26 is in the wall of the active-site cleft. Its replacement with serine results in the rearrangement of nearby residues, most notably Tyr 18, suggesting that the increase in stability may result from the removal of strain. Thr 151 in the wild-type structure is far from the active site and appears to sterically prevent the access of solvent to a preformed binding site. In the mutant, the removal of the methyl group allows access to the solvent binding site and, in addition, the Ser 151 hydroxyl rotates to a new position so that it also contributes to solvent binding. Residue 93 is in a highly exposed site on the surface of the molecule, and presumably is equally solvent exposed in the unfolded protein. It is, therefore, not surprising that the substitution Ala 93-->Thr does not change stability. The mutant structures show how chemically similar mutations can have different effects on both the structure and stability of the protein, depending on the structural context. The results also illustrate the power of random mutagenesis in obtaining variants with a desired phenotype.(ABSTRACT TRUNCATED AT 250 WORDS)

Carnitine palmitoyltransferase (CPT) II deficiency is the most common inherited disorder of lipid metabolism affecting skeletal muscle. We have identified a missense mutation (Ser113Leu) in one patient with the classical muscular symptomatology. Transfection experiments in COS cells demonstrate that the mutation drastically depresses the catalytic activity of CPT II. The mutation results in normal synthesis but a markedly reduced steady-state level of the protein, indicating decreased stability of mutant CPT II. The Ser113Leu mutation is the most frequent cause of CPT II deficiency. The mutation can be detected easily by restriction analysis enabling molecular diagnosis of most patients and identification of heterozygous carriers.

The effects of engineered disulfide bonds on autodigestion and thermostability of Bacillus subtilis neutral protease (NP-sub) were studied using site-directed mutagenesis. After modelling studies two locations that might be capable of forming disulfide bonds, both near previously determined autodigestion sites in NP-sub, were selected for the introduction of cysteines. Analysis of mutant enzymes showed that disulfide bonds were indeed formed in vivo, and that the mutant enzymes were fully active. The introduced disulfides did not alter the autodigestion pattern of the NP-sub. All mutant NP-subs exhibited decreased thermostability, which, by using reducing agents, was shown to be caused by the introduction of the cysteines and not by the formation of the disulfides. Mutants containing one cysteine exhibited intermolecular disulfide formation at elevated temperatures, which, however, was shown not to be the cause of the decreased thermostability. Combining the present data with literature data, it would seem that the introduction of disulfide bridges is unsuitable for the stabilization of proteases. Possible explanations for this phenomenon are discussed.

Sau3 A and Hind III restriction fragments of Clostridium cylindrosporum genomic DNA were used to isolate clones containing 80% of the N10-H4folate synthetase gene in a 5' fragment and the remaining 20% of the gene in the 3' fragment. These fragments were joined at a common SnaB I restriction site and expressed in Escherichia coli at a level equivalent to what is normally found in C. cylindrosporum. Sequence comparisons show a large degree of homology with genes from two other clostridial species, including a thermophile. Certain conserved sequences found in the three clostridial proteins and in the N10-H4folate synthetase portion of eukaryotic C1-H4folate synthases may represent consensus sequences for nucleotide and H4folate binding.

Systematic replacement of the amino acid residues in Escherichia coli ribonuclease HI with those in the thermophilic counterpart has revealed that two mutations, His62-->Pro (H62P) and Lys95-->Gly (K95G), increased the thermostability of the protein. These single-site mutant proteins, together with the mutant proteins His62-->Ala (H62A), Lys95-->Asn (K95N) and Lys95-->Ala (K95A), were crystallized and their structures were determined at 1.8 A resolution. The crystal structures of these mutant proteins reveal that only the local structure around each mutation site is essential for the increase in thermostability. For each mutant protein, the stabilization mechanism is considered to be as follows: (i) H62P is stabilized because of a decrease in the entropy of the unfolded state, without a change in the native backbone structure; (ii) K95G is stabilized since the strain caused by the left-handed backbone structure in the typical 3:5 type loop is eliminated; and (iii) K95N is slightly stabilized by a hydrogen bond formed between the side-chain N delta-atom of the mutated aspargine residue and the main-chain carbonyl oxygen within the same residue.

The evolutionally conserved aspartyl residues (Asp57, Asp98 and Asp152) in human glutathione S-transferase P1-1 were replaced with alanine by site-directed mutagenesis to obtain the mutants (D57A, D98A and D152A). The replacement of Asp98 with alanine resulted in a decrease of the affinity for S-hexyl-GSH-agarose, a 5.5-fold increase of the KmGSH and a 2.9-fold increase of the I50 of S-hexyl-GSH for GSH-CDNB conjugation. Asp98 seems to participate in the binding of GSH through hydrogen bonding with the alpha-carboxylate of the gamma-glutamyl residue of GSH. The kcat of D98A was 2.6-fold smaller than that of the wild-type, and the pKa of the thiol group of GSH bound in D98A was approximately 0.8 pK units higher than those in the wild-type. Asp98 also seems to contribute to the activation of GSH to some extent. On the other hand, most of the kinetic parameters of D57A and D152A were similar to those of the wild-type. However, the thermostabilities of D57A and D152A were significantly lower than that of the wild-type. Asp57 and Asp152 seem to be important for maintaining the proper conformation of the enzyme.

Dihydrofolate reductase mutants with amino acid replacements in the active center (Thr35-->Asp mutant, Arg57-->His mutant and the mutant with triple replacement Thr35-->Asp, Asn37-->Ser, Arg57-->His) were obtained by site-directed mutagenesis. The stabilization effect of trimethoprim and NADP.H on the protein tertiary structure in vitro has been investigated. In the case of mutants with a 'weak' tertiary structure (Thr35-->Asp35 and the triple mutant) the separate addition of ligands does not affect their stability. The simultaneous addition of these ligands to Thr35-->Asp35 and the triple mutant leads to the large increase in their stability. A distinct correlation was found between the in vitro studied stability of the mutant proteins to the urea- or heat-induced denaturation and the level of proteolytic degradation of these mutants previously observed in vivo.

On the basis of three-dimensional information, many amino acid substitutions were introduced in the thermostable neutral protease (NprM) of Bacillus stearothermophilus MK232 by site-directed mutagenesis. When Glu at position 143 (Glu-143), which is one of the proposed active sites, was substituted for by Gln and Asp, the proteolytic activity disappeared. F114A (Phe-114 to Ala), Y110W (Tyr-110 to Trp), and Y211W (Tyr-211 to Trp) mutant enzymes had higher activity (1.3- to 1.6-fold) than the wild-type enzyme. When an autolysis site, Tyr-93, was replaced by Gly and Ser, the remaining activities of those mutant enzymes were higher than that of the wild-type enzyme.

A 10 residue beta-hairpin, which is characteristic of thermostable Bacillus neutral proteases, was engineered into the thermolabile neutral protease of Bacillus subtilis. The recipient enzyme remained fully active after introduction of the loop. However, the mutant protein exhibited autocatalytic nicking and a 0.4 degree C decrease in thermostability. Two additional point mutations designed to improve the interactions between the enzyme surface and the introduced beta-hairpin resulted in reduced nicking and increased thermostability. After the introduction of both additional mutations in the loop-containing mutant, nicking was largely prevented and an increase in thermostability of 1.1 degrees C was achieved.

The 247-260 and 289-299 alpha-helices of Bacillus subtilis neutral protease have a lysine in their N-terminal turn. These lysines were replaced by Ser or Asp in order to improve electrostatic interactions with the alpha-helix dipole. After replacing Lys by Ser at positions 249 or 290, the thermostability of the enzyme was increased by 0.3 and 1.0 degrees C, respectively. The Asp249 and Asp290 mutants exhibited a stabilization of 0.6 and 1.2 degrees C, respectively. The results show the feasibility of stabilizing enzymes by introducing favourable residues at the end of alpha-helices.

The engineered disulfide bridge between residues 21 and 142 of phage T4 lysozyme spans the active-site cleft and can be used as a switch to control the activity of the enzyme (Matsumura, M. & Matthews, B.W., 1989, Science 243, 792-794). In the oxidized form the disulfide increases the melting temperature of the protein by 11 degrees C at pH 2. The crystal structure of this mutant lysozyme has been determined in both the reduced and oxidized forms. In the reduced form, the crystal structure of the mutant is shown to be extremely similar to that of wild type. In the oxidized form, however, the formation of the disulfide bridge causes the alpha-carbons of Cys 21 and Cys 142, on opposite sides of the active-site cleft, to move toward each other by 2.5 A. In association with this movement, the amino-terminal domain of the protein undergoes a rigid-body rotation of 5.1 degrees relative to the carboxy-terminal domain. This rotation occurs about an axis passing through the junction of the amino-terminal and carboxy-terminal domains and is also close to the axis that best fits the apparent thermal motion of the amino-terminal domain seen previously in crystals of wild-type lysozyme. Even though the engineered Cys 21-Cys 142 disulfide links together the amino-terminal and carboxy-terminal domains of T4 lysozyme, it does not reduce the apparent mobility of the one domain relative to the other. The pronounced "hinge-bending" mobility of the amino-terminal domain that is suggested by the crystallographic thermal parameters of wild-type lysozyme persists in the oxidized (and reduced) mutant structures. In the immediate vicinity of the introduced disulfide bridge the mutant structure is more mobile (or disordered) than wild type, so much so that the exact conformation of Cys 21 remains obscure. As with the previously described disulfide bridge between residues 9 and 164 of T4 lysozyme (Pjura, P.E., Matsumura, M., Wozniak, J.A., & Matthews, B.W., 1990, Biochemistry 29, 2592-2598), the engineered cross-link substantially enhances the stability of the protein without making the folded structure more rigid.

Six mutant xylanases were obtained by in vitro mutagenesis of a xylanase gene from the extremely thermophilic bacterium Caldocellum saccharolyticum. The temperature stability of all enzymes was affected by mutation to various degrees and one of the xylanases had an altered temperature optimum. The mutations had no effect on the pH optimum. The C. saccharolyticum xylanase showed strong homology to several thermophilic and mesophilic xylanases, and comparison of primary sequences allowed the localization of probable active sites and residues involved in thermostability.

Alcohol oxidase of methylotrophic yeast is an FAD-containing enzyme. When in its active form, the enzyme is an octamer and located in the peroxisomes. To study the importance of FAD-binding on the activity, octamerization and intracellular localization of the enzyme, alcohol oxidase of Hansenula polymorpha was mutated in its presumed nucleotide-binding domain, which is formed by the N-terminal sequence. Whereas mutations of a glutamic acid residue (E42) reduced the stability of the octamer, it hardly affected enzyme activity and expression. However, replacements of three conserved glycines (G13, G15 and G18) and a conserved glutamic acid (E39) within the fold had severe effects. The mutations not only resulted in loss of enzyme activity but in reduced protein levels as well, probably due to decreased stability of the mutant alcohol oxidase. However, octamerization of the protein still occurred. The existence of inactive octameric proteins provides information about the formation pathway of this octameric flavoprotein.

The expression of kanamycin nucleotidyltransferase (KNTase) in Escherichia coli results in different forms of the protein, depending on the temperature; soluble active enzyme is synthesized at 23 degrees C but the protein is mostly aggregated and inactive in inclusion bodies when made at 37 degrees C. However, active enzyme can be recovered by solubilization of the inclusion bodies with 8 M urea followed by dilution of the denaturant, indicating that the polypeptide is not damaged covalently but is present in a misfolded state. The availability of thermostable mutants of KNTase allows a test of the hypothesis that formation of inclusion bodies when proteins are highly expressed in E. coli is due to the accumulation of a folding intermediate that is prone to temperature-dependent aggregation. Because these mutants were isolated by cloning the KNTase gene into the thermophile Bacillus stearothermophilus and selecting for kanamycin resistance at high growth temperatures, they must be thermostable for both synthesis and activity and must have folding intermediates that are less susceptible to the formation of aggregates. Indeed, whereas decreasing the temperature from 37 to 23 degrees C increased the KNTase specific activity 10-fold in cells expressing the wild-type enzyme, this change resulted in only a 2.1-fold increase for the TK1 (Asp80----Tyr) mutant and a 1.7-fold increase for the TK101 (Asp80----Tyr and Thr130----Lys) double mutant. The strategy of cloning in thermophiles and selecting or screening for mutants that fold correctly to yield biological activity at high growth temperatures may be useful in overcoming the problem of the insolubility of some proteins when expressed in heterologous hosts.

This study was undertaken to characterize the effect of three point mutations made on aprA-subtilisin on the stability of the protein to both heat- and detergent-induced denaturation. Asparagine residues at positions 109 and 218 were replaced with serine residues to prevent the possible cyclization between these asparagines and the adjacent glycine residues and hence to increase the long-term stability. The effect of these substitutions on conformational stability was examined by thermal denaturation. At high calcium concentrations, the Ser109-substituted analog showed a 3 degrees C higher transition temperature than that of aprA-subtilisin, while the Ser218 substituted analog had a 4 degrees C higher transition temperature. The analog with both changes had a 7 degrees C higher transition temperature than that of the original aprA-subtilisin, indicating that the contributions of the individual mutations were additive. The analog with both mutations also exhibited increased stability in the presence of sodium dodecyl sulfate (SDS) when compared to aprA-subtilisin. In addition to the above two mutations, the asparagine at position 76, located in the high affinity Ca(2+) binding loop of subtilisin, was changed to aspartic acid. The effect of this mutation on the thermal stability of the protein was examined at different calcium concentrations. The analog with all three mutations exhibited little dependence on calcium concentration below 1 mM levels, while the proteins without the mutation at asparagine-76 displayed a strong dependence of melting temperature on Ca(2+) concentration in this range. At much higher calcium concentrations, the analog with three mutations showed an increase in stability similar to that observed with aprA-subtilisin. The analog with three mutations also exhibited greater stability to SDS-induced denaturation than both aprA-subtilisin and the Ser109- and Ser218-substituted analogs. The activation energy barrier for loss of structure in 1% SDS for the analog with all three mutations was increased over that for aprA-subtilisin by 16 kcal/ml. These results suggest that the mutation of asparagine-76 to aspartic acid increases the affinity of the primary Ca(2+) binding site.

Although the structural gene for human dopamine-beta-hydroxylase (DBH) has been cloned, the mechanism by which DBH physical properties and activity are regulated is not well understood. Previous reports have suggested that three-allele or two-locus models may account for the genetic regulation of these traits in human blood. It is an interesting challenge to determine the extent to which quantitative analyses will complement or guide molecular genetic studies. In this study we analyzed data on the physical property of DBH thermal stability and DBH activity in 230 individuals in 53 families in an attempt to clarify genetic mechanisms for the inheritance of these traits. Commingling and segregation analyses of the thermal stability data provided the first clear evidence of a major gene polymorphism for DBH thermal stability analyzed as a quantitative trait. Major gene transmission was supported within a mixed model (chi 2[3] = 13.39, P less than .004). In keeping with earlier findings, similar analyses of DBH activity provided strong evidence of genetic transmission. However, in our data support for a major gene polymorphism was equivocal (chi 2(2) = 2.99, P = .22).