Plasma paraoxonase polymorphism: a new enzyme assay, population, family, biochemical, and linkage studies.

Plasma paraoxonase hydrolyzes paraoxon, the principal metabolite of the insecticide parathione. A genetic polymorphism for enzyme activity has been previously demonstrated. We describe a new assay based on the differential inhibition by EDTA of plasma paraoxonase from persons with the high-activity allele (PX*H) that suggests a trimodality of activity levels in population studies. The gene frequency of the low activity allele (PX*L) in 531 Seattle blood donors of European origin was .7207. Family studies were consistent with codominant autosomal inheritance of two alleles, PX*L (low) and PX*H (high), coding for products with different activity levels. Biochemical measurements of sera from presumed homozygotes for the two different alleles revealed minor physicochemical differences suggestive of a structural difference between the allelic products. No evidence for linkage of the paraoxonase locus with any of 19 polymorphic markers would be detected.     

Model involving gene inactivation in the generation of autosomal recessive mutants in mammalian cells in culture.

We present evidence for a two-step model for expression of the recessive phenotype at the diploid adenine phosphoribosyl transferase (aprt) locus in Chinese hamster ovary cells. This model proposes a high-frequency event leading to allelic inactivation and a low-frequency event leading to a structural alteration of the APRT protein. Either event can occur first, resulting in two types of heterozygous cells. The proposed model is based on analysis of Chinese hamster ovary presumptive aprt heterozygotes and APRT- mutants, derived by two different laboratories. The major class of heterozygotes (class 1) had approximately 50% parental APRT activity, 50% immunologically precipitable APRT protein, and only wild-type enzyme as based on two-dimensional gel electrophoresis and thermal inactivation studies. We propose that one allele at the aprt locus has been inactivated in these heterozygotes. APRT- mutants derived from any single class 1 heterozygote arose at a low frequency and contained either no immunologically detectable APRT protein or an APRT enzyme which was, in most cases, demonstrably altered. The second class of heterozygotes, consisting of two independent isolates, gave rise to APRT- cells at a high frequency (10(-3) to 10(-5). These heterozygous cell lines had 50% of parental APRT activity and only wild-type spot, or wild-type and an electrophoretic variant spot, on two-dimensional gels. These aprt heterozygotes appear to have arisen by mutation at one allele. APRT- mutants derived from either heterozygote of this class had all lost the wild-type activity, consistent with the proposed model.     

Contribution of arylsulfatase A mutations located on the same allele to enzyme activity reduction and metachromatic leukodystrophy severity

The occurrence of different mutations on the same arylsulfatase A allele is not uncommon, due to the high frequency of several variants, among which the pseudodeficiency mutations are particularly important. We identified a late infantile metachromatic leukodystrophy patient carrying on one allele the new E253K mutation and the known T391S polymorphism, and on the other allele the common P426L mutation, usually associated with the adult or juvenile form of the disease, and the N350S and *96A>G pseudodeficiency mutations. To analyze the contribution of mutations located on the same allele to enzyme activity reduction, as well as the possible phenotype implications, we performed transient expression experiments using arylsulfatase A cDNAs carrying the identified mutations separately and in combination. Our results indicate that mutants containing multiple mutations cause a greater reduction of ARSA activity than do the corresponding single mutants, the total deficiency likely corresponding to the sum of the reductions attributed to each mutation. Consequently, each mutation may contribute to ARSA activity reduction, and, therefore, to the degree of disease severity. This is particularly important for the alleles containing a disease-causing mutation and the pseudodeficiency mutations: in these alleles pseudodeficiency could play a role in affecting the clinical phenotype.     

Enzymatic activity of naturally occurring 1-acylglycerol-3-phosphate-O-acyltransferase 2 mutants associated with congenital generalized lipodystrophy

Mutations in the gene encoding 1-acylglycerol-3-phosphate-O-acyltransferase 2 (AGPAT2) have been reported in patients with congenital generalized lipodystrophy (CGL). AGPAT2, a 278 amino acid protein, belongs to the acyltransferase enzyme family, and has two conserved motifs, NHX(4)D and EGTR, involved in the enzymatic activity. The AGPATs catalyze acylation of lysophosphatidic acid (LPA) to phosphatidic acid (PA) during the biosynthesis of glycerophospholipids and triglycerides from glycerol-3-phosphate. The present studies were designed to determine the enzymatic activity of AGPAT2 mutants found in CGL patients to provide a molecular explanation for the phenotype and to obtain additional information about the structure-function relationship of AGPAT2 protein. The enzymatic activities of the wild type AGPAT2 and mutants were determined in cell lysates of overexpressing Chinese hamster ovary cells by measuring the conversion of [(3)H]LPA to [(3)H]PA in the presence of oleoyl-coenzyme A. Whereas, the R68X, 221delGT, 252delMRT, D180fsX251, and V167fsX183 mutants had markedly reduced enzymatic activity (median <15% of the wild type), the mutants, 140delF, G136R, and L228P, retained median activity ranging from 15% to 40% of the wild type enzyme. However, the missense mutant, A239V, had 90% of the wild type activity. We suggest that reduction in AGPAT2 enzymatic activity underlies the loss of adipose tissue in CGL. Our observations reveal an important role of various carboxy-terminal residues in determining the enzymatic activity of AGPAT2.     

Murine "housekeeping" enzyme (genetic locus: Idh-1) is regulated in an allele-specific manner

The murine "housekeeping" enzyme, cytosolic NADP-isocitrate dehydrogenase (E.C.1.1.1.42) (genetic locus: Idh-1), exhibited a complex pattern of allele-specific expression. Protein electrophoresis on cellulose-acetate gels and determination of relative enzymatic activity by means of densitometry revealed that in heart tissue (but not liver tissue) of certain hybrid crosses the AA-homodimer was underrepresented relative to total enzymatic activity, and the degree of underrepresentation changed during development. In mixtures of homozygous tissue extracts of heart tissue (but not liver tissue) the AA-homodimer was underrepresented relative to the BB-homodimer. Relative activity of allelic isozymes varied as a function of tissue (heart versus liver), age, and the parental source of the Idh-1 alpha allele, but did not vary as a function of sex. Allele-specific expression was also exhibited in kidney tissue of the same animals. In adult male kidney tissue extracts from heterozygotes, the AA-homodimer was underrepresented relative to total enzymatic activity; in adult female kidney tissue extracts from heterozygotes, a more codominant phenotype was observed. Tissue extracts from immature hybrid animals exhibited a phenotype midway between the adult male and adult female phenotypes. Tissue extracts from castrated males exhibited a phenotype equivalent to that seen in females. Relative activity of allelic isozymes in kidney varied as a function of age and sex, but did not vary as a function of the parental source of the Idh-1 alpha allele. While cytosolic NADP-IDH is a "housekeeping" enzyme, expressed in multiple tissues of the mouse, differences in the relative intensities of allelic isozyme bands provide evidence for tissue- and stage-specific regulatory variation.     

Hysteretic properties of soluble F1 ATPase from Escherichia coli. II. Nucleotide effects on the slow changes of the enzyme kinetic behaviour.

In a previous paper from this laboratory it was shown that EC.F₁ ATPase exhibits a temperature dependent transition between two stable states, called L (low activity) and H (high activity). They differ three-fold in specific activity. I report here the effects of ADP and ATP on this transition. Both nucleotides were found to shift the equilibrium between the two states in the direction of the L state. Further, the velocity of conversion from one state to the other was accelerated by the presence of the nucleotides. It was shown that the two states of the enzyme exhibit different kinetic properties in that: (i) the L state gives a hyperbolic curve when specific activity is plotted against substrate concentration, and ADP produces an immediate competitive inhibition and (ii) the H state exhibits negative cooperativity when such a curve is plotted, and shows a delayed competitive inhibition by ADP. Furthermore, when enzyme in the H state is loaded with ATP its complex kinetic behavior disappears; ADP does not have this effect. The data are interpreted to mean that the H state depleted of ATP may act as an enzyme bearing two alternative catalytic sites.     

Low β-glucuronidase enzyme activity and mutations in the human β-glucuronidase gene in mild mucopolysaccharidosis type VII, pseudodeficiency and a heterozygote

Deficiency of β-glucuronidase is the cause of the human lysosomal storage disorder mucopolysaccharidosis type VII (MPS VII). The wide interfamilial variation in the presentation of this disorder complicates clinical diagnosis. Since greatly reduced β-glucuronidase enzyme activity may also be found in healthy individuals (pseudodeficiency), diagnosis based on the biochemical phenotype is also difficult. This is illustrated by the patients studied here, who had extremely mild symptoms confined to the spine, or tachycardia, or upper respiratory infection, and who had low β-glucuronidase activity, and excessive granulation of granulocytes and monocytes on routine blood smears. Low enzyme activity was caused by mutations in the β-glucuronidase gene in all cases. One patient was homozygous for the previously described D152N allele. Family information and ³⁵SO₄-uptake studies clearly demonstrated that he was pseudodeficient, with symptoms unrelated to his low β-glucuronidase activity. Two patients of another family were compound heterozygotes for a C38G and a Y626H allele, and were probably extremely mild MPS VII patients. The low β-glucuronidase activity in another mild MPS VII patient was due to reduced biosynthesis of stable mRNA from one allele, and a W446X mutation on the second. Extremely low β-glucuronidase enzyme activity was also found in the serum of a carrier of a 1801ΔT allele, possibly as a consequence of a dominant-negative effect. A combination of investigations is necessary in order to differentiate between mild disease and pseudodeficiency in individuals with enzyme activities close to the threshold. Received: 31 May 1997 / Accepted: 26 Augsut 1997     

Disease-associated mutations inactivate AMP-lysine hydrolase activity of Aprataxin

Ataxia-oculomotor apraxia syndrome 1 is an early onset cerebellar ataxia that results from loss of function mutations in the APTX gene, encoding Aprataxin, which contains three conserved domains. The forkhead-associated domain of Aprataxin mediates protein-protein interactions with molecules that respond to DNA damage, but the cellular phenotype of the disease does not appear to be consistent with a major loss in DNA damage responses. Disease-associated mutations in Aprataxin target a histidine triad domain that is similar to Hint, a universally conserved AMP-lysine hydrolase, or truncate the protein NH2-terminal to a zinc finger. With novel fluorigenic substrates, we demonstrate that Aprataxin possesses an active-site-dependent AMP-lysine and GMP-lysine hydrolase activity that depends additionally on the zinc finger for protein stability and on the forkhead associated domain for enzymatic activity. Alleles carrying any of eight recessive mutations associated with ataxia and oculomotor apraxia encode proteins with huge losses in protein stability and enzymatic activity, consistent with a null phenotype. The mild presentation allele, APTX-K197Q, associated with ataxia but not oculomotor apraxia, encodes a protein with a mild defect in stability and activity, while enzyme encoded by the atypical presentation allele, APTX-R199H, retained substantial function, consistent with altered and not loss of activity. The data suggest that the essential function of Aprataxin is reversal of nucleotidylylated protein modifications, that all three domains contribute to formation of a stable enzyme, and that the in vitro behavior of cloned APTX alleles can score disease-associated mutations.     

Stage-specific expression of Caenorhabditis elegans ribonuclease H1 enzymes with different substrate specificities and bivalent cation requirements

Ribonuclease H1 (RNase H1) is a widespread enzyme found in a range of organisms from viruses to humans. It is capable of degrading the RNA moiety of DNA-RNA hybrids and requires a bivalent ion for activity. In contrast with most eukaryotes, which have one gene encoding RNase H1, the activity of which depends on Mg(2+) ions, Caenorhabditis elegans has four RNase H1-related genes, and one of them has an isoform produced by alternative splicing. However, little is known about the enzymatic features of the proteins encoded by these genes. To determine the differences between these enzymes, we compared the expression patterns of each RNase H1-related gene throughout the development of the nematode and the RNase H activities of their recombinant proteins. We found gene-specific expression patterns and different enzymatic features. In particular, besides the enzyme that displays the highest activity in the presence of Mg(2+) ions, C. elegans has another enzyme that shows preference for Mn(2+) ion as a cofactor. We characterized this Mn(2+)-dependent RNase H1 for the first time in eukaryotes. These results suggest that there are at least two types of RNase H1 in C. elegans depending on the developmental stage of the organism.     

Genetic control of the natural killer cell activity in SJL and other strains of mice

Murine natural killer (NK) cell activity against lymphoma targets can be classified into three major functional phenotypes, i.e., low, inducible, and high, according to the levels of endogeneous activity and the extent of augmentation by interferon (IFN) or IFN inducers, as previously described. The prototype strains identifying these three phenotypes are SJL, A.SW, and B10.S, respectively, all bearing the H-2s haplotype. In the present study, the genetic basis of the low phenotype of SJL mice was examined further. F1 hybrid offspring of crosses between SJL and a strain with the high NK phenotype (B10.S, B10.D2, B10, C3H/HeN, or D1.LP) invariably expressed the high NK phenotype, indicating recessiveness of the low phenotype. Crosses between SJL and another low NK strain, such as A/J, A/HeN, or I/St, resulted in offspring of either the inducible or the high NK phenotype. Such genetic complementation between the low NK pairs indicates that the low phenotype of SJL and that of the other strains have different genetic bases. F1 hybrid mice between SJL and an inducible strain, A/WySn, were inducible, but those between SJL and the second inducible strain, A.SW, had the high NK phenotype. Thus, the congenic A/WySn and A.SW have distinct genotypes resulting in the same inducible phenotype. According to analyses of the segregating offspring from backcrosses of (SJL X B10.S)F1 mice to SJL, a single gene difference is responsible for the low endogenous level of NK activity in SJL as opposed to the high endogenous level in B10.S, and that the difference in three genes accounts for the poor responsiveness of NK cells to IFN in SJL mice. Studies of the two congenic lines of SJL, i.e., SJL-Igha and SJL-nu, indicated that the Igh locus is irrelevant for the low NK phenotype of SJL, but the nu locus clearly is relevant; SJL mice homozygous for the nu allele were phenotypically inducible in contrast to the nu/+ or +/+ mice which are low. The nu gene homozygosity rendered SJL mice responsive to IFN, not only in NK activity against lymphomas but also in ADCC activity against antibody-coated lymphoma cells.     

Identification and expression of five mutations in the human acid sphingomyelinase gene causing types A and B Niemann-Pick disease. Molecular evidence for genetic heterogeneity in the neuronopathic and non-neuronopathic forms.

The deficient activity of the human lysosomal hydrolase, acid sphingomyelinase (ASM, EC 3.1.4.12), results in the neuronopathic (Type A) and non-neuronopathic (Type B) forms of Niemann-Pick disease (NPD). To investigate the genetic basis of the phenotypic heterogeneity in NPD, the molecular lesions in the ASM gene were determined from three unrelated NPD patients and evaluated by transient expression in COS-1 cells. A Type A NPD patient of Asian Indian ancestry (proband 1) was homoallelic for a T to A transversion in exon 2 of the ASM gene which predicted a premature stop at codon 261 of the ASM polypeptide (designated L261X). In contrast, an unrelated Type A patient of European ancestry (proband 2) was heteroallelic for a two-base (TT) deletion in exon 2 which caused a frame-shift mutation at ASM codon 178 (designated fsL178), leading to a premature stop at codon 190, and a G to A transition in exon 3 which caused a methionine to isoleucine substitution at codon 382 (designated M382I). Transient expression of the fsL178, L261X, and M382I mutations in COS-1 cells demonstrated that these lesions did not produce catalytically active ASM, consistent with the severe neuronopathic Type A NPD phenotype. In contrast, an unrelated Type B patient of European descent (proband 3) was heteroallelic for two missense mutations, a G to A transition in exon 2 which predicted a glycine to arginine substitution at ASM codon 242 (designated G242R), and an A to G transition in exon 3 which resulted in an asparagine to serine substitution at codon 383 (designated N383S). Interestingly, the G242R allele produced ASM activity in COS-1 cells at levels about 40% of that expressed by the normal allele, thereby explaining the mild Type B phenotype of proband 3 and the high residual activity (i.e. approximately 15% of normal) in cultured lymphoblasts. In contrast, the N383S allele did not produce catalytically active enzyme. None of these five ASM mutations was detected in over 60 other unrelated NPD patients analyzed, nor were these mutations found in over 100 normal ASM alleles. Thus, small deletions or nonsense mutations which trunctated the ASM polypeptide, or missense mutations that rendered the enzyme noncatalytic, resulted in Type A NPD disease, whereas a missense mutation that produced a defective enzyme with residual catalytic activity caused the milder nonneuronopathic Type B phenotype. These findings have facilitated genotype/phenotype correlations for this lysosomal storage disease and provided insights into the functional organization of the ASM polypeptide.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

亮白曲霉乳糖酶基因在毕赤酵母中的高效分泌表达及酶学性质研究

将克隆的亮白曲霉 (Aspergilluscandidus)乳糖酶基因lacb′插入到毕赤酵母 (Pichiapastoris)高效表达载体pPIC9中 ,与分泌信号肽序列α_因子融合 ,通过同源重组将lacb′整合到酵母染色体上。通过SDS_PAGE检测和表达产物的酶活性筛选 ,得到重组转化子 ,证明乳糖酶获得有效分泌和高效表达。表达的乳糖酶为糖蛋白 ,表观分子量为 130kD ,脱糖基后的蛋白分子量下降为 110kD。经过 5L小罐高密度发酵 ,重组酵母中酶蛋白表达量为 6mg mL发酵液 ,每毫升发酵液中乳糖酶的活力为 36 0 0U ,高于目前国内外报道的水平。进一步研究了表达产物的酶学性质 ,该酶最适pH为 5 . 2 ,最适反应温度 6 0℃ ,比活性为 70 6 . 5± 2 . 6U mg ,Km为 1 7mmol L ,Vmax为 3. 3μmol min。与米曲霉ATCC 2 0 4 2 3的乳糖酶相比 ,该乳糖酶具热稳定性强、比活性高、pH范围宽等特点。     

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.     

Cloning and DNA sequence analysis of an X-prolyl dipeptidyl aminopeptidase gene from Lactococcus lactis subsp. lactis NCDO 763.

Lactococcus lactis subsp. lactis NCDO 763 (also designated ML3) possesses an X-prolyl dipeptidyl aminopeptidase (X-PDAP; EC 3.4.14.5). X-PDAP mutants were selected by an enzymatic plate assay on the basis of their inability to hydrolyze an L-phenylalanyl-L-proline-beta-naphthylamide substrate. A DNA bank from L. lactis subsp. lactis NCDO 763 was constructed in one of these X-PDAP mutants, and one clone in which the original X-PDAP phenotype was restored was detected by the enzymatic plate assay. The X-PDAP gene, designated pepXP, was further subcloned and sequenced. It codes for a protein containing 763 residues. Comparison of the amino-terminal sequence of the X-PDAP enzyme with the amino acid sequence deduced from the pepXP gene indicated that the enzyme is not subjected to posttranslational modification or exported via processing of a signal peptide. The pepXP gene from L. lactis subsp. lactis NCDO 763 in more than 99% homologous to the pepXP gene from L. lactis subsp. cremoris P8-2-47 described elsewhere (B. Mayo, J. Kok, K. Venema, W. Bockelmann, M. Teuber, H. Reinke, and G. Venema, Appl. Environ. Microbiol. 57:38-44, 1991) and is also conserved in other lactococcal strains.     

Mutational analysis in longest known survivor of mucopolysaccharidosis type VII

Mucopolysaccharidosis VII (MPS VII) is an autosomal recessive disorder caused by the deficiency of beta-glucuronidase leading to the intralysosomal storage of heparan, dermatan, and chondroitin sulfate. Here, we report the identification of two novel missense mutations K350N and R577L in a 37-year-old patient with beta-glucuronidase deficiency and a relatively mild MPS VII phenotype. Expression of the K350N mutation in baby hamster kidney cells has revealed residual enzymatic activity and normal transport of the enzyme to the lysosome. However, expression of the R577L or the double mutant K350N/R577L results in rapid degradation of the enzyme in early biosynthetic compartments and a total loss of enzymatic activity. We attribute the mild phenotype to the residual catalytic activity provided by the K350N mutant. At the time of her death at the age of 37 years, this patient was the longest known survivor with MPS VII.     

Glucose phosphate isomerase deficiency with hereditary hemolytic anemia in a Spanish family: clinical and familial studies.

A new case of glucose phosphate isomerase deficiency associated with cogenital nonspherocytic hemolytic anemia is described in a 12-year-old girl of Spanish origin. The parents exhibited erythrocyte glucose phosphate isomerase activity between 50 and 60% of normal. The enzyme of the propositus had normal Michaelis-Menten constants both for F-6-P and G-6-P, but abnormal pH optimum and decreased heat stability at 48 degrees C. On starch-gel electrophoresis the father's enzyme was normal but the mother's showed a cathodic migrating band in addition to the normal one. The enzyme from the propositus exhibited only one band with cathodal mobility of 116% of the main band found in normal subjects. It is postulated that the propositus is double heterozygous for two abnormal alleles, and the mother contributes a mutant allele with abnormal electrophoretic mobility and thermolability at 48 degrees C whereas the father contributes an allele without enzymatic activity.     

Identification of critical residues in the propeptide of LasA protease of Pseudomonas aeruginosa involved in the formation of a stable mature protease

LasA protease is a 20-kDa elastolytic and staphylolytic enzyme secreted by Pseudomonas aeruginosa. LasA is synthesized as a preproenzyme that undergoes proteolysis to remove a 22-kDa amino-terminal propeptide. Like the propeptides of other bacterial proteases, the LasA propeptide may act as an intramolecular chaperone that correctly folds the mature domain into an active protease. To locate regions of functional importance within proLasA, linker-scanning insertional mutagenesis was employed using a plasmid containing lasA as the target. Among the 5 missense insertions found in the mature domain of proLasA, all abolished enzymatic activity but not secretion. In general, the propeptide domain was more tolerant to insertions. However, insertions within a 9-amino-acid region in the propeptide caused dramatic reductions in LasA enzymatic activity. All mutant proLasA proteins were still secreted, but extracellular stability was low due to clustered insertions within the propeptide. The codons of 16 residues within and surrounding the identified 9-amino-acid region were subjected to site-directed mutagenesis. Among the alanine substitutions in the propeptide that had a major effect on extracellular LasA activity, two (L92A and W95A) resulted in highly unstable proteins that were susceptible to proteolytic degradation and three (H94A, I101A, and N102A) were moderately unstable and allowed the production of a LasA protein with low enzymatic activity. These data suggest that these clustered residues in the propeptide may play an important role in promoting the correct protein conformation of the mature LasA protease domain.     

Effects of Y132H and F145L substitutions on the activity, azole resistance and spectral properties of Candida albicans sterol 14-demethylase P450 (CYP51): a live example showing the selection of altered P450 through interaction with environmental compounds

Three variants of Candida albicans CYP51 (sterol 14-demethylase P450) having Y132H and/or F145L substitutions were purified and characterized to reveal the effects of these amino acid substitutions on the enzymatic properties and azole resistance of the enzyme. Y132H and F145L substitutions modified the spectral properties of the enzyme, suggesting that they caused some structural change modifying the heme environments of CYP51. Y132H and F145L substitutions increased the resistance of the enzyme to azole compounds but considerably decreased the catalytic activity. This fact represents a trade-off between acquisition of azole resistance and maintenance of high activity in the CYP51 having Y132H and F145L substitutions. A fluconazole-resistant C. albicans strain DUMC136 isolated from patients receiving long-term azole treatment was a homozygote of the altered CYP51 having Y132H and F145L substitutions. However, neither of these substitutions was found in CYP51 of wild-type C. albicans so far studied. These facts suggest that the azole-resistant variant having Y132H and/or F145L substitutions might be selected only under azole-rich environments because of its azole resistance and impaired catalytic activity. This may be a live example showing one of the important processes of P450 diversification, the selection of altered P450 through the interaction with environmental compounds.