Characterization of the mutant N-acetylglucosaminylphosphotransferase in I-cell disease and pseudo-Hurler polydystrophy: complementation analysis and kinetic studies

Complementation was examined among various types of I-cell disease and pseudo-Hurler polydystrophy by monitoring N-acetylglucosaminylphosphotransferase activity in multinucleated cells produced by fusing pair combinations of cultured skin fibroblasts. Patients with the classical forms of these disorders (5 I-cell disease and 3 pseudo-Hurler polydystrophy cell lines) comprised one complementation group and 5 cell lines from patients with variant forms of pseudo-Hurler polydystrophy comprised a distinct complementation group. In the first group, total or partial deficiency of the transferase activity was demonstrated with both natural (lysosomal enzymes) and artificial (alpha-methylmannoside) acceptor substrates with low Vmax but apparently normal Km values for the donor (UDP-GlcNAc) and acceptor (alpha-methylmannoside) substrates. The activity toward artificial substrate could be inhibited by adding exogenous lysosomal enzyme preparations to the reaction mixture. In the second group, the cells demonstrated deficiency of the transferase activity toward lysosomal enzyme acceptors but had normal activity toward alpha-methylmannoside acceptor and this activity could not be inhibited by the addition of exogenous lysosomal enzyme preparations. These findings suggest that N-acetylglucosaminylphosphotransferase is composed of at least two distinct subunits, a catalytic subunit which is absent or defective in the first complementation group, and a recognition subunit which is altered or deficient in the second group.     

Mucolipidoses II and III variants with normal N-acetylglucosamine 1-phosphotransferase activity toward alpha-methylmannoside are due to nonallelic mutations.

Normal N-acetylglucosamine 1-phosphotransferase activity toward mono- and oligosaccharide acceptor substrates was detected in cultured skin fibroblasts from mucolipidoses II and III patients who were designated as variants (one of four mucolipidosis II and three out of six mucolipidosis III patients examined). The activity toward natural lysosomal protein acceptors was absent or deficient in cell preparations from all patients with classical as well as variant forms of mucolipidoses II and III. Complementation analysis, using fused and cocultivated mutant fibroblast combinations, revealed that, while cell lines with variant mucolipidosis III constituted a complementation group distinct from that of classical forms of mucolipidoses II and III, the variant mucolipidosis II cell line belonged to the same complementation group as did the classical forms. In contrast to the mutant enzyme from variant mucolipidosis III patients that failed to recognize lysosomal proteins as the specific acceptor substrates, the activity toward alpha-methylmannoside in the variant mucolipidosis II patient could be inhibited by exogenous lysosomal enzyme preparations (bovine beta-glucuronidase and human hexosaminidase A). These findings suggest that N-acetylglucosamine 1-phosphotransferase is composed of at least two distinct polypeptides: (1) a recognition subunit that is defective in the mucolipidosis III variants and (2) a catalytic subunit that is deficient or altered in the classical forms of mucolipidoses II and III as well as in the mucolipidosis II variant.     

Radiometric assays of N-acetylglucosaminylphosphotransferase and alpha-N-acetylglucosaminyl phosphodiesterase with substrates labeled in the glucosamine moiety

The assay of fibroblast and leukocyte-N-acetylglucosaminylphosphotransferase with alpha-methylmannoside acceptor and commercially available UDP-[3H or 14C]N-acetylglucosamine donor was modified to yield low background and consequently high sensitivity and reliability comparable to those obtained with the synthetically made [beta-32P]UDP-N-acetylglucosamine donor. This was achieved by an additional elution step that removed free [3H or 14C]N-acetylglucosamine which appeared to be the breakdown product responsible for the high background. In addition, the [3H or 14C]N-acetylglucosamine-1-phospho-6-alpha-methylmannoside product of the transfer reaction was then isolated and, following desalting, could serve as a substrate for the assay of alpha-N-acetylglucosaminyl phosphodiesterase. Cell preparations of patients with I-cell disease and pseudo-Hurler polydystrophy demonstrated severe to moderate deficiency of transferase activity and normal phosphodiesterase activity toward the respective substrates labeled with 3H or 14C in the glucosamine moiety.     

Light and heavy lysosomes: characterization of N-acetyl-{beta}-D-hexosaminidase isolated from normal and I-cell disease lymphoblasts

We previously reported that I-cell disease lymphoblasts maintainnormal or near-normal intracellular levels of lysosomal enzymes,even though N-acetylglucosamine-1-phosphotransferase activityis severely depressed or absent (Little et al., Biochem. J.,248, 151–159, 1987). The present study, employing subcellularfractionation on colloidal silica gradients, indicates thatboth light and heavy lysosomes isolated from I-cell diseaseand pseudo-Hurler polydystrophy lymphoblasts possess normalspecific activity levels of N-acetyl-ß-D-hexosaminidase,-D-mannosidase and ß-D-glucuronidase. These currentfindings are in contrast to those of cultured fibroblasts fromthe same patients, where decreased intralysosomal enzyme activitiesare found. Column chromatography on Ricinus communis revealedthat N-acetyl-ß-D-hexosaminidase in both heavy andlight I-cell disease lysosomal fractions from lymphoblasts possessesan increased number of accessible galactose residues (30–50%)as compared to the enzyme from the corresponding normal controls.Endo-ß-N-acetylglucos-aminidase H treatment of N-acetyl-ß-D-hexosaminidasefrom the I-cell lysosomal fractions suggests that the majorityof newly synthesized high-mannose-type oligosaccharide chainsare modified to complex-type carbohydrates prior to being transportedto lysosomes. This result from lymphoblasts differs from previousfindings with fibroblasts, where N-acetyl-ß-D-hexosaminidasefrom I-cell disease and pseudo-Hurler polydystrophy lysosomesexhibited properties associated with predominantly high-mannose-typeoligosaccharide chains. The current results imply that differentcell types may modify the carbohydrate side chains of lysosomalenzymes in a differential manner, and that selected cell typesmay also employ mechanisms other than the mannose-6-phosphatepathway for targeting lysosomal enzymes to lysosomes. I-cell disease lymphoblasts lysosomes mannose-6-phosphate oligosaccharide chains pseudo-Hurler polydystrophy     

Demonstration of the heterozygous state for I-cell disease and pseudo-Hurler polydystrophy by assay of N-acetylglucosaminylphosphotransferase in white blood cells and fibroblasts

The biochemical abnormalities of I-cell disease (mucolipidosis II) and pseudo-Hurler polydystrophy (mucolipidosis III) can be explained by a deficiency of the enzyme UDP-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase. We demonstrate here that obligate heterozygotes for these autosomal recessive diseases have intermediate levels of this enzymatic activity in homogenates of peripheral blood white cells and in extracts from cultured fibroblasts. This finding provides further evidence that the enzyme deficiency is the primary genetic defect in these diseases. In addition, the previous observation that obligate heterozygotes for mucolipidosis III have elevations of total serum beta-hexosaminidase outside the range of normal was confirmed. In studies of three pedigrees of patients with mucolipidosis III, these techniques were used to score individuals at risk for the carrier state.     

Phosphorylation and subcellular location of {alpha}-L-fucosidase in Iymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

Mannose 6-phosphate is a recognition marker used by many newlymade acid hydrolases for their transport to lyso-somes. Previously,we investigated the incorporation of ³²Pi into -L-fucosidaseof lymphoid cell lines from a healthy individual (control) andan I-cell disease patient [DiCioccio and Miller, Glycobiology,1, 595–604 (1991)]. Phosphoserine was found in immunoprecipitable-L-fucosidase of both control and I-cell lymphoid cells, butmannose 6-phosphate was identified only in enzyme of controlcells. Extension of this investigation to lymphoid culturesof a pseudo-Hurler polydystrophy patient also identified onlyphosphoserine in -L-fucosidase. Using [³H] mannose instead of³²Pi, the precise identification of mannose 6-phosphate in -L-fucosidaseof control cells, and its absence in -L-fucosidase of I-celland pseudo-Hurler cells, was established. The stoichiometryof phosphorylation of -L-fucosidase in I-cell, pseudo-Hurlerand control lymphoid cells was 3, 4 and 10 mol Pi/mol enzyme,respectively. -L-Fucosidase was located in lysosomes isolatedfrom control, I-cell and pseudo-Hurler lymphoid cells by subcelluarfractionation on Percoll density gradients. Both I-cell andpseudo-Hurler lymphoid cells displayed normal intralysosomalactivity of -L-fucosidase despite lack of the mannose 6-phosphatemarker. Thus, I-cell and pseudo-Hurler lymphoid cells must possessa mannose 6-phosphate-independent mechanism for directing -L-fucosidaseto lysosomes. Phosphorylation of -L-fucosidase in pseudo-Hurlerand I-cell lymphoid cultures was found almost exclusively inintracellular and not in extracellular enzyme, suggesting thatphosphoserine may participate in the localization of -L-fucosidasein lysosomes of these cells. -L-fucosidase I-cell disease lysosome phosphorylation pseudo-Hurler polydystrophy     

Mucolipidosis II (I-cell disease) and mucolipidosis IIIA (classical pseudo-hurler polydystrophy) are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene

Mucolipidosis II (MLII; I-cell disease) and mucolipidosis IIIA (MLIIIA; classical pseudo-Hurler polydystrophy) are diseases in which the activity of the uridine diphosphate (UDP)-N-acetylglucosamine:lysosomal enzyme N-acetylglucosamine-1-phosphotransferase (GlcNAc-phosphotransferase) is absent or reduced, respectively. In the absence of mannose phosphorylation, trafficking of lysosomal hydrolases to the lysosome is impaired. In these diseases, mistargeted lysosomal hydrolases are secreted into the blood, resulting in lysosomal deficiency of many hydrolases and a storage-disease phenotype. To determine whether these diseases are caused by mutations in the GlcNAc-phosphotransferase alpha / beta -subunits precursor gene (GNPTAB), we sequenced GNPTAB exons and flanking intronic sequences and measured GlcNAc-phosphotransferase activity in patient fibroblasts. We identified 15 different mutations in GNPTAB from 18 pedigrees with MLII or MLIIIA and demonstrated that these two diseases are allelic. Mutations in both alleles were identified in each case, which demonstrated that GNPTAB mutations are the cause of both diseases. Some pedigrees had identical mutations. One frameshift mutation (truncation at amino acid 1171) predominated and was found in both MLII and MLIIIA. This mutation was found in combination with severe mutations (i.e., mutations preventing the generation of active enzyme) in MLII and with mild mutations (i.e., mutations allowing the generation of active enzyme) in MLIIIA. Some cases of MLII and MLIIIA were the result of mutations that cause aberrant splicing. Substitutions were inside the invariant splice-site sequence in MLII and were outside it in MLIIIA. When the mutations were analyzed along with GlcNAc-phosphotransferase activity, it was possible to confidently distinguish these two clinically related but distinct diseases. We propose criteria for distinguishing these two disorders by a combination of mutation detection and GlcNAc-phosphotransferase activity determination.     

I-Cell disease: isoelectric focusing, concanavalin A-Sepharose 4B binding and kinetic properties of human liver acid beta-D-galactosidases.

Isoelectric focusing of the acid beta-D-galactosidases (beta-D-galactoside galactohydrolase, EC 3.2.1.23) in normal crude liver supernatant fluids demonstrated multiple isoelectric forms in the pH range 4.58-5.15, while corresponding I-cell disease samples showed an absence of isoelectric forms in the pH range 4.99-5.15. Concanavalin A-Sepharose 4B chromatography of the I-cell disease mutant C.A. demonstrated a 31% and 37% decrease in the binding of 4-methyl-umbelliferyl-beta-D-galactosidase and GM1 beta-D-galactosidase activities, respectively, when compared to normal samples. Isoelectric focusing profiles of the concanavalin A-Sepharose 4B alpha-methyl-D-mannoside effluents containing normal and I-cell disease acid beta-D-galactosidase were generally similar, but the unadsorbed I-cell disease enzyme from concanavalin A-Sepharose 4B demonstrated more activity in the pH range 4.21-4.49 than normals. Normal and I-cell disease acid beta-D-galactosidase "A" and "B", separated by gel column chromatography were found to have similar properties with respect to apparent molecular weights pH vs. activity profiles and apparent Km values for the 4 methylumbelliferyl-beta-D-galactopyranoside, GM1-ganglioside and asialofetuin (ASF) substrates. However, the apparent V values for the ICD samples were consistently reduced when compared to the results obtained with the corresponding normal fractions. The greatest decreases in apparent V were obtained for acid beta-D-galactosidase activities in I-cell disease crude supernatant fluids, and for the separated I-cell disease "B" enzyme. The differences in the isoelectric focusing profiles, the altered binding to concanavalin A-Sepharose 4B, and the reduced V values with natural and synthetic substrates may be related to changes in carbohydrate composition of I-cell disease acid beta-D-galactosidase.     

Studies of the synthesis,structure and function of the phosphorylated oligosaccharides of lysosomal enzymes

Newly synthesized lysosomal enzymes were found to contain N-acetylglucosamine residues in phosphodiester linkage to the 6 position of the mannose residues on high-mannose type oligosaccharides. The formation of these structures was shown to be catalyzed by a specific N-acetylglucosaminylphosphotransferase enzyme, that utilises UDP-N-acetylglucosamine as a donor. The phosphorylation reaction can take place on any of four or five positions on the high-mannose oligosaccharide. Subsequently an α-N-acetylglucosaminylphosphodiesterase removes the outer blocking N-acetylglucosamine residues to generate the mature phosphomannsoyl recognition signal. This signal is responsible for the targetting of newly synthesized lysosomal enzymes to lysosomes. The human syndromes of I-cell disease (Mucolipidosis II) and pseudo-Hurler polydystrophy (Mucolipidosis III) were shown to be caused by deficiency of the first enzyme in the pathway, the UDP-N-acetylglucosamine: Glycoprotein N-acetylglucosaminylphosphotransferase.     

Impaired cholesterol esterification in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy

Cholesterol esterification was examined in cultured skin fibroblasts from patients with I-cell disease and pseudo-Hurler polydystrophy by incubating cells pretreated without fetal calf serum for 48h, with (14C) cholesterol for 24h. Impaired cholesterol esterification was found in these cells and free cholesterol was accumulated in plasma membrane and Golgi fractions. This impairment was also induced in control cells by adding leupeptin (20 micrograms/ml) or monensin (2 micrograms/ml). These findings suggest the importance of the role of lysosomes for esterification of cholesterol and give a hint as to the basic defect in type C Niemann-Pick disease.     

Preliminary evidence for a processing error in the biosynthesis of Gaucher activator in mucolipidosis disease types II and III.

Activator protein (AP), which stimulated fibroblast sphingomyelinase activity, was isolated from the spleen of a patient with Gaucher's disease type I by the combined techniques of heat and alcohol denaturation, DEAE-cellulose column chromatography, gel filtration, preparative polyacrylamide-gel electrophoresis and decyl-agarose chromatography. Urea/sodium dodecyl sulphate (SDS)/polyacrylamide-gel electrophoresis showed two bands, one with an Mr of approx. 3,000 and the other with an Mr of 5,000-6,500. Similarly, SDS/polyacrylamide-gel electrophoresis performed in the absence of urea revealed the presence of two components, one of which adsorbed to a concanavalin A (Con A) column. Both components stimulated sphingomyelinase activity. On a non-denaturing polyacrylamide gel containing Triton X-100, four major components, two of which bound to Con A, were detected with the dye Stains-All. Cross-reacting material (CRM) to polyclonal Gaucher spleen AP antibodies was detected in normal fibroblasts and in fibroblasts from patients with sphingomyelinase and beta-glucocerebrosidase deficiency states (Niemann-Pick and Gaucher's diseases respectively). CRM in normal fibroblasts adsorbed to Con A columns and had the same mobility on SDS/polyacrylamide-gel electrophoresis as Con A-adsorbing Gaucher spleen AP. Normal AP was not observed in mucolipidosis type II (I-cell disease) fibroblasts; instead, extracts from these cells revealed the presence of two closely migrating bands with higher Mr values than normal fibroblast CRM. Furthermore, extracts of media from I-cell fibroblast cultures, but not from control or Gaucher fibroblast cultures, contained AP activity towards sphingomyelinase and beta-glucocerebrosidase. Fibroblasts from a patient with mucolipidosis type III (pseudo-Hurler polydystrophy) showed an intermediate pattern consisting of normal as well as the higher-Mr CRM. Our data provide evidence for the existence of AP in cultured skin fibroblasts and suggest that these proteins may be targetted to the lysosome by post-translational modification in a similar manner to that reported for lysosomal enzymes.     

Characterization of beta-D-galactosidase isolated from I-cell disease liver.

The residual beta-D-galactosidase activity (10% of normal) present in an autopsy sample of liver derived from an I-cell patient has been characterized. The pH optima for both I-cell and normal acid 4-methylumbelliferyl beta-D-galactoside activities were 4.35. The adsorption and elution profiles of the I-cell enzyme from Con A-Sepharose were similar to those of normal liver beta-D-galactosidase. Although starch gel electrophoresis revealed the presence of beta-D-galactosidase A and B in I-cell disease liver, the A band was more diffuse and migrated less anodally than the A band from normal liver. The electrophoretic mobilities of both I-cell and normal beta-D-galactosidase A appeared to decrease after treatment with neuraminidase. Kinetic studies of the I-cell and normal level beta-D-galactosidase demonstrated similar apparent Km values with respect to the 4-methylumbelliferyl beta-D-galactoside and Gm1 ganglioside, whereas the Vmax values obtained for the I-cell enzyme were 10- to 12-fold lower than those of the normal enzyme for both substrates.     

Biosynthesis, processing, and secretion of {alpha}-L-fucosidase in lymphoid cells from patients with I-cell disease and pseudo-Hurler polydystrophy

N-Acetylglucosamine 1-phosphotransferase is a key enzyme requiredfor synthesis of the mannose 6-phosphate recognition markerthat is used by many newly made acid hydrolases for their transportto lysosomes. It has previously been found that lymphoid cellsfrom patients with I-cell disease and pseudo-Hurler polydystrophyhave nearly normal intracellular and intralysosomal activitiesof several lysosomal acid hydrolases, despite a deficiency ofN-acetylglucosamine 1-phosphotransferase. These results suggestthat lymphoid cells may provide an important system to investigatealternate mechanisms for targeting newly made acid hydrolasesto lysosomes. In the present study, the biosynthesis, processingand secretion of -L-fucosidase in I-cell and pseudoHurler lymphoidcells was used as a model system to study the existence of suchmechanisms. The level of intracellular -L-fucosidase proteinin exponentially growing I-cell or pseudo-Hurler lymphoid cultureswas statistically indistinguishable from the mean of 19 controlcultures. A 1.5 h [³⁵S]methionine pulse experiment showed that-L-fucosidase is initially sythesized by I-cell, pseudo-Hurlerand control cultures as an intracellular form (M_r = 58 000).Companion cultures chased with methionine from 2 to 21 h processedthe enzyme to an intracellular form (M_r = 60 000) and an extracellularform (M_r = 62 000). All enzyme forms were glycoproteins withpolypeptide chains of Mr 52 000. In control cells incubatedwith radioactive inorganic phosphate (³²Pi), <1% of the ³²Piincorporated into -L-fucosidase was associated with carbohydratechains and >99% with polypeptide chains. In I-cell diseaselymphoid cells, the ³²Pi incorporated into -L-fucosidase wasassociated solely with polypeptide chains. A qualitative analysisof phosphorylated residues identified phosphoserine in -L-fucosidasefrom control and I-cell lymphoid cells. Only -L-fucosidase fromcontrol cells contained mannose 6-phosphate. These results areconsistent with the proposal that I-cell lymphoid cells mayuse a mannose 6-phosphate-independent mechanism for routing-L-fucosidase. Additional metabolic labelling experiments demonstratedthe presence of ³²P-labelled -L-fucosidase in both cells andmedium of a control lymphoid culture, but only in cells of anI-cell lymphoid culture. In contrast, -L-fucosidase labelledwith [³⁵S]methionine was found in cells and medium of controland I-cell lymphoid cultures. Since phosphoserine was only foundto occur in intracellular, but not in extracellular -L-fucosidaseof the I-cell culture, we speculate that phosphoserine may beinvolved in intracellular retention of -L-fucosidase in I-celllymphoid cells. -L-fucosidase I-cell disease lymphoid cells phos-phorylation pseudo-Hurler polydystrophy     

Defective phosphorylation and processing of beta-hexosaminidase by intact cultured fibroblasts from patients with mucolipidosis III

Previous studies of the synthesis, phosphorylation, and processing of β-hexosaminidase in cultured fibroblasts from normal individuals and from patients with mucolipidosis II (I-cell disease) (A. Hasilik and E. F. Neufeld, 1980, J. Biol. Chem.225, 4937–4946) have been extended to fibroblasts derived from patients with a related genetic disorder, mucolipidosis III (pseudo-Hurler polydystrophy). The enzyme was biosynthetically labeled in pulse-chase experiments with [³H]leucine and ³³P_i, and isolated from cells and medium by immunoprecipitation. The constitutent α and β chains of the enzyme were separated by polyacrylamide gel electrophoresis under reducing and denaturing conditions, visualized by autoradiography and fluorography, extracted from the gel, and quantitated by liquid scintillation spectrometry. Enzyme produced by fibroblasts from mucolipidosis III patients had a very low but detectable phosphate content; a high proportion of newly made enzyme was secreted, though some remained within the cells and was processed to mature enzyme; the presence of NH₄Cl during the labeling and chase did not significantly increase the amount of enzyme secreted. The β-hexosaminidase produced by mucolipidosis III fibroblasts thus resembled more closely that produced by fibroblasts from patients with mucolipidosis II than the normal enzyme. β-Hexosaminidase made by fibroblasts from mucolipidosis II heterozygotes was similar to the normal enzyme with respect to phosphorylation, processing, and secretion. Mucolipidosis II and III fibroblasts could endocytose normal precursor β-hexosaminidase and process it to the mature form. The deficiency of mature enzyme in the patients' cells may therefore be attributed to failure of the unphosphorylated enzyme to be incorporated into lysosomes, where processing would normally occur.     

A cluster of missense mutations at Arg356 of human steroid 21-hydroxylase may impair redox partner interaction

Lesions in the gene encoding steroid 21-hydroxylase result in congenital adrenal hyperplasia, with impaired secretion of cortisol and aldosterone from the adrenal cortex and overproduction of androgens. A limited number of mutations account for the majority of mutated alleles, but additional rare mutations are responsible for the symptoms in some patients. A total of 11 missense mutations has previously been implicated in this enzyme deficiency. We describe two novel missense mutations, both affecting the same amino acid residue, Arg356. The two mutations, R356P and R356Q, were reconstructed by in vitro site-directed mutagenesis, the proteins were transiently expressed in COS-1 cells, and enzyme activity towards the two natural substrates, 17-hydroxyprogesterone and progesterone, was determined. The R356P mutant reduced enzyme activity to 0.15% towards both substrates, whereas the R356Q mutant exhibited 0.65% of normal activity towards 17-hydroxyprogesterone, and 1.1% of normal activity towards progesterone. These activities correspond to the degrees of disease manifestation of the patients in whom they were found. Arg356 is located in a region which recently has been implicated in redox partner interaction, by modelling the structure of two other members of the cytochrome P450 superfamily. Of the 11 previously described missense mutations, three affect arginine residues within this protein domain. With the addition of R356P and R356Q, there is a clear clustering of five mutations to three closely located basic amino acids. This supports the model in which this protein domain is involved in redox partner interaction, which takes places through electrostatic interactions between charged amino acid residues. Received:17 December 1996 / Revised: 28 January 1997     

Mutation of amino acids in the alpha 1,3-fucosyltransferase motif affects enzyme activity and Km for donor and acceptor substrates

Alpha 1,3-fucosyltransferases (FucT) share a conserved amino acid sequence designated the alpha 1,3 FucT motif that has been proposed to be important for nucleotide sugar binding. To evaluate the importance of the amino acids in this motif, each of the alpha 1,3 FucT motif amino acids was replaced with alanine (alanine scanning mutagenesis) in human FucT VI, and the resulting mutant proteins were analyzed for enzyme activity and kinetically characterized in those cases in which the mutant protein had sufficient activity. Two of the mutant proteins were inactive, six had less than 1% of wild-type activity, and four had approximately 10-50% of wild-type enzyme activity. Three of the mutant proteins with significant enzyme activity had substantially larger Km (5 to 15 times) for GDP-fucose than FucT VI wild-type enzyme. The fourth mutant protein with significant enzyme activity (S249A) had a Km at least 10 times larger than wild-type FucT VI for the acceptor substrate, with only a slightly larger (2-3 times) Km for GDP-fucose. Thus mutation of any of the amino acids within the alpha 1,3 FucT motif to Ala affects alpha 1,3-FucT activity, and substitution of Ala for some of the alpha 1,3 FucT motif amino acids results in proteins with altered kinetic constants for both the acceptor and donor substrates. Secondary structure prediction suggests a helix-loop-helix fold for the alpha 1,3 FucT motif, which can be used to rationalize the effects of mutations in terms of 3D structure.     

G6PD Huntsville: a new glucose-6-phosphate dehydrogenase associated with chronic hemolytic anemia

Summary We describe a previously unreported glucose-6-phosphate dehydrogenase (G6PD) variant. G6PD Huntsville was found in a Caucasian male, resident of Huntsville, Alabama who was investigated for otherwise unexplained chronic hemolytic anemia. An unusual feature of this unique, apparently hemolytic, G6PD mutant is that its red cell enzymatic activity has not been decreased. The mutant enzyme is unstable. Additionally, the enzyme variant is characterized by normal electrophoretic mobility, biphasic and slightly alkaline pH optimum, and abnormal kinetics for the natural substrates G6PD and NADP as well as the artificial substrates deamino NADP. Its activity for another artificial substrate 2-deoxy G6PD is normal. The inhibition constant for NADPH is normal. The subject has had no evidence of episodic jaundice.     

Directed evolution of transketolase substrate specificity towards an aliphatic aldehyde

Mutants of transketolase (TK) with improved substrate specificity towards the non-natural aliphatic aldehyde substrate propionaldehyde have been obtained by directed evolution. We used the same active-site targeted saturation mutagenesis libraries from which we previously identified mutants with improved activity towards glycolaldehyde, which is C2-hydroxylated like all natural TK substrates. Comparison of the new mutants to those obtained previously reveals distinctly different subsets of enzyme active-site mutations with either improved overall enzyme activity, or improved specificity towards either the C2-hydroxylated or non-natural aliphatic aldehyde substrate. While mutation of phylogenetically variant residues was found previously to yield improved enzyme activity on glycolaldehyde, we show here that these mutants in fact gave improved activity on both substrate types. In comparison, the new mutants were obtained at conserved residues which interact with the C2-hydroxyl group of natural substrates, and gave up to 5-fold improvement in specific activity and 64-fold improvement in specificity towards propionaldehyde relative to glycolaldehyde. This suggests that saturation mutagenesis can be more selectively guided for evolution towards either natural or non-natural substrates, using both structural and sequence information.     

Yersiniabactin synthetase: probing the recognition of carrier protein domains by the catalytic heterocyclization domains, Cy1 and Cy2, in the chain-initiating HWMP2 subunit

The HMWP2 subunit of yersiniabactin (Ybt) synthetase, a 230 kDa nonribosomal peptide synthetase (NRPS) making the N-terminus of the Ybt siderophore of Yersinia pestis, has one cysteine-specific adenylation (A) domain, three carrier protein domains (ArCP, PCP1, PCP2), and two heterocyclization domains (Cy1, Cy2). The A domain loads the two PCP domains with cysteines that get heterocyclized by the Cy domains to yield a tricyclic hydroxyphenylthiazolinylthiazolinyl (HPTT) chain lodged in thioester linkage to the PCP2 domain. The interdomain recognition by the Cy1 and Cy2 domains for the three carrier proteins was tested using inactivating mutations at the conserved serine that is phosphopantetheinylated in each carrier domain (S52A, S1439A, and S1977A). These mutant forms of HMWP2 were tested for in trans complementation by carrier protein fragments: holo-ArCPs (S52A), holo-PCP1 and analogues (S1439A), and holo-PCP2 and analogues (S1977A). The S52A mutant tests the recognition of the Cy1 domain for donor acyl-ArCP substrates, while the S1439A mutant tests the specificity of the same Cy1 domain for downstream substrates presented by distinct PCPs. The S1439A likewise tests the recognition of Cy2 for its upstream PCP-tethered acyl donor. The S1977A mutant analogously tests the Cy2 domain for downstream Cys-PCP recognition. In all cases in trans complementation was successful with the carrier protein fragments, allowing kinetic probes of catalytic efficiency for PCP scaffolds and for uncoupling of the condensation and heterocyclization functions of Cy1 and Cy2. Overall, the Cy domains tested showed a definite selectivity for the upstream protein scaffold but were more relaxed toward the downstream acceptor protein. This work points to the importance of protein-protein interactions in mediating directional chain growth in NRPS and presents the first systematic exploration of how the protein scaffolds affect catalytic efficiency.     

Construction of cellobiose phosphorylase variants with broadened acceptor specificity towards anomerically substituted glucosides

The general application of glycoside phosphorylases such as cellobiose phosphorylase (CP) for glycoside synthesis is hindered by their relatively narrow substrate specificity. We have previously reported on the creation of Cellulomonas uda CP enzyme variants with either modified donor or acceptor specificity. Remarkably, in this study it was found that the donor mutant also displays broadened acceptor specificity towards several β‐glucosides. Triple mutants containing donor (T508I/N667A) as well as acceptor mutations (E649C or E649G) also display a broader acceptor specificity than any of the parent enzymes. Moreover, further broadening of the acceptor specificity has been achieved by site‐saturation mutagenesis of residues near the active site entrance. The best enzyme variant contains the additional N156D and N163D mutations and is active towards various alkyl β‐glucosides, methyl α‐glucoside and cellobiose. In comparison with the wild‐type C. uda CP enzyme, which cannot accept anomerically substituted glucosides at all, the obtained increase in substrate specificity is significant. The described CP enzyme variants should be useful for the synthesis of cellobiosides and other glycosides with prebiotic and pharmaceutical properties. Biotechnol. Bioeng. 2010;107: 413–420. © 2010 Wiley Periodicals, Inc.