Characterization of two mutations associated with epimerase-deficiency galactosemia, by use of a yeast expression system for human UDP-galactose-4-epimerase.

UDP-galactose-4-epimerase (GALE) is a highly conserved enzyme that catalyzes the interconversion of UDP-galactose and UDP-glucose. Impairment of this enzyme in humans results in one of two clinically distinct forms of epimerase-deficiency galactosemia-one benign, the other severe. The molecular and biochemical distinction between these disorders remains unknown. To enable structural and functional studies of both wild-type and patient-derived alleles of human GALE (hGALE), we have developed and applied a null-background yeast expression system for the human enzyme. We have demonstrated that wild-type hGALE sequences phenotypically complement a yeast gal10 deletion, and we have biochemically characterized the wild-type human enzyme isolated from these cells. Furthermore, we have expressed and characterized two mutant alleles, L183P-hGALE and N34S-hGALE, both derived from a patient with no detectable GALE activity in red blood cells but with approximately 14% activity in cultured lymphoblasts. Analyses of crude extracts of yeast expressing L183P-hGALE demonstrated 4% wild-type activity and 6% wild-type abundance. Extracts of yeast expressing N34S-hGALE demonstrated approximately 70% wild-type activity and normal abundance. However, yeast coexpressing both L183P-hGALE and N34S-hGALE exhibited only approximately 7% wild-type levels of activity, thereby confirming the functional impact of both substitutions and raising the intriguing possibility that some form of dominant-negative interaction may exist between the mutant alleles found in this patient. The results reported here establish the utility of the yeast-based hGALE-expression system and set the stage for more-detailed studies of this important enzyme and its role in epimerase-deficiency galactosemia.     

The conserved tyrosine residues 401 and 1044 in ATP sites of human P-glycoprotein are critical for ATP binding and hydrolysis: evidence for a conserved subdomain, the A-loop in the ATP-binding cassette

Each nucleotide-binding domain (NBD) of mammalian P-glycoproteins (Pgps) and human ATP-binding cassette (ABC) B subfamily members contains a tyrosine residue approximately 25 residues upstream of the Walker A domain. To assess the role of the conserved Y401 and Y1044 residues of human Pgp, we substituted these residues with F, W, C, or A either singly or together. The mutant proteins were expressed in a Vaccinia virus-based transient expression system as well as in baculovirus-infected HighFive insect cells. The Y401F, Y401W, Y1044F, Y1044W, or Y401F/Y1004F mutants transported fluorescent substrates similar to the wild-type protein. On the other hand, Y401L and Y401C exhibited partial (30-50%) function, and transport was completely abolished in Y401A, Y1044A, and Y401A/Y1044A mutant Pgps. Similarly, in Y401A, Y1044A, and Y401A/Y1044A mutants, TNP-ATP binding, vanadate-induced trapping of nucleotide, and ATP hydrolysis were completely abolished. Thus, an aromatic residue upstream of the Walker A motif in ABC transporters is critical for binding of ATP. Additionally, the crystal structures of several NBDs in the nucleotide-bound form, data mining, and alignment of 18,514 ABC domains with the consensus conserved sequence in a database of all nonredundant proteins indicate that an aromatic residue is highly conserved in approximately 85% of ABC proteins. Although the role of this aromatic residue has previously been studied in a few ABC proteins, we provide evidence for a near-universal structural and functional role for this residue and recognize its presence as a conserved subdomain approximately 25 amino acids upstream of the Walker A motif that is critical for ATP binding. We named this subdomain the "A-loop" (aromatic residue interacting with the adenine ring of ATP).     

Alternative proton donors/acceptors in the catalytic mechanism of the glutathione reductase of Escherichia coli: the role of histidine-439 and tyrosine-99

The cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase was placed under the control of the tac promoter in the plasmid pKK223-3, allowing expression of glutathione reductase at levels approximately 40,000 times those of untransformed cells. This greatly facilitated purification of the enzyme. By directed mutagenesis of the gor gene, His-439 was changed to glutamine (H439Q) and alanine (H439A). The tyrosine residue at position 99 was changed to phenylalanine (Y99F), and in another experiment, the H439Q and Y99F mutations were united to form the double mutant Y99FH439Q. His-439 is thought to act in the catalytic mechanism as a proton donor/acceptor in the glutathione-binding pocket. The H439Q and H439A mutants retain approximately 1% and approximately 0.3%, respectively, of the catalytic activity of the wild-type enzyme. This reinforces our previous finding [Berry et al. (1989) Biochemistry 28, 1264-1269] that direct protonation and deprotonation of the histidine residue are not essential for the reaction to occur. The retention of catalytic activity by the H439A mutant demonstrates further that a side chain capable of hydrogen bonding to a water molecule, which might then act as proton donor, also is not essential at this position. Tyr-99 is a further possible proton donor in the glutathione-binding pocket, but the Y99F mutant was essentially fully active, and the Y99FH439Q double mutant also retained approximately 1% of the wild-type specific activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Conserved F84 and P86 residues in alphaB-crystallin are essential to effectively prevent the aggregation of substrate proteins

Previously, we have shown that residues 73-92 (sequence DRFSVNLDVKHFSPEELKVK) in alphaB-crystallin are involved in preventing the formation of light scattering aggregates by substrate proteins. In this study, we made single substitutions of three conserved amino acid residues (H83 --> A, F84 --> G, and P86 --> A) and a nonconserved amino acid residue (K90 --> C) in the functional region of alphaB-crystallin and evaluated their role in anti-aggregation activity. Mutation of conserved residues led to changes in intrinsic tryptophan intensity, bis-ANS binding, and in the secondary and tertiary structures. The H83A mutation led to a twofold increase in molar mass, while the other mutants did not produce significant changes in the molar mass when compared to that of wild-type protein. The chaperone-like activity of the H83A mutant was enhanced by 15%-20%, and the chaperone-like activity of F84G and P86A mutants was reduced by 50%-65% when compared to the chaperone-like activity of wild-type alphaB-crystallin. The substitution of the nonconserved residue (K90 --> C) did not induce an appreciable change in the structure and function of the mutant protein. Fluorescence resonance energy transfer (FRET) assay demonstrated that destabilized ADH interacted near the K90 region in alphaB-crystallin. The data show that F84 and P86 residues are essential for alphaB-crystallin to effectively prevent the aggregation of substrate proteins. This study further supports the involvement of the residues in the 73-92 region of alphaB-crystallin in substrate protein binding and chaperone-like action.     

Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase

UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.     

Nitration of tyrosine 92 mediates the activation of rat microsomal glutathione s-transferase by peroxynitrite

There is increasing evidence that protein function can be modified by nitration of tyrosine residue(s), a reaction catalyzed by proteins with peroxidase activity, or that occurs by interaction with peroxynitrite, a highly reactive oxidant formed by the reaction of nitric oxide with superoxide. Although there are numerous reports describing loss of function after treatment of proteins with peroxynitrite, we recently demonstrated that the microsomal glutathione S-transferase 1 is activated rather than inactivated by peroxynitrite and suggested that this could be attributed to nitration of tyrosine residues rather than to other effects of peroxynitrite. In this report, the nitrated tyrosine residues of peroxynitrite-treated microsomal glutathione S-transferase 1 were characterized by mass spectrometry and their functional significance determined. Of the seven tyrosine residues present in the protein, only those at positions 92 and 153 were nitrated after treatment with peroxynitrite. Three mutants (Y92F, Y153F, and Y92F, Y153F) were created using site-directed mutagenesis and expressed in LLC-PK1 cells. Treatment of the microsomal fractions of these cells with peroxynitrite resulted in an approximately 2-fold increase in enzyme activity in cells expressing the wild type microsomal glutathione S-transferase 1 or the Y153F mutant, whereas the enzyme activity of Y92F and double site mutant was unaffected. These results indicate that activation of microsomal glutathione S-transferase 1 by peroxynitrite is mediated by nitration of tyrosine residue 92 and represents one of the few examples in which a gain in function has been associated with nitration of a specific tyrosine residue.     

Tyrosine residue 300 is important for activity and stability of branching enzyme from Escherichia coli

Branching enzyme belongs to the alpha-amylase family, which includes enzymes that catalyze hydrolysis or transglycosylation at alpha-(1,4)- or alpha-(1,6)-glucosidic linkages. In the alpha-amylase family, four highly conserved regions are proposed to make up the active site. From amino acid sequence analysis a tyrosine residue is completely conserved in the alpha-amylase family. In Escherichia coli branching enzyme, this residue (Y300) is located prior to the conserved region 1. Site-directed mutagenesis of the Y300 residue in E. coli branching enzyme was used in order to study its possible function in branching enzymes. Replacement of Y300 with Ala, Asp, Leu, Ser, and Trp resulted in mutant enzymes with less than 1% of wild-type activity. A Y300F substitution retained 25% of wild-type activity. Kinetic analysis of Y300F showed no effect on the Km value. The heat stability of Y300F was analyzed, and this was lowered significantly compared to that of the wild-type enzyme. Y300F also showed lower relative activity at elevated temperatures compared to wild-type. Thus, these results show that Tyr residue 300 in E. coli branching enzyme is important for activity and thermostability of the enzyme.     

Dissection of the two transferase activities of the Pasteurella multocida hyaluronan synthase: two active sites exist in one polypeptide

Type A Pasteurella multocida, an animal pathogen, employs a hyaluronan [HA] capsule to avoid host defenses. PmHAS, the 972-residue membrane-associated hyaluronan synthase, catalyzes the transfer of both GlcNAc and GlcUA to form the HA polymer. To define the catalytic and membrane-associated domains, pmHAS mutants were analyzed. PmHAS1-703 is a soluble, active HA synthase suggesting that the carboxyl-terminus is involved in membrane association of the native enzyme. PmHAS1-650 is inactive as a HA synthase, but retains GlcNAc-transferase activity. Within the pmHAS sequence, there is a duplicated domain containing a short motif, Asp-Gly-Ser, that is conserved among many beta-glycosyltransferases. Changing this aspartate in either domain to asparagine, glutamate, or lysine reduced the HA synthase activity to low levels. The mutants substituted at residue 196 possessed GlcUA-transferase activity while those substituted at residue 477 possessed GlcNAc-transferase activity. The Michaelis constants of the functional transferase activity of the various mutants, a measure of the apparent affinity of the enzymes for the precursors, were similar to wild-type values. Furthermore, mixing D196N and D477K mutant proteins in the same reaction allowed HA polymerization at levels similar to the wild-type enzyme. These results provide the first direct evidence that the synthase polypeptide utilizes two separate glycosyltransferase sites.     

Deletion of N-terminal amino acids from human lecithin:cholesterol acyltransferase differentially affects enzyme activity toward alpha- and beta-substrate lipoproteins

Lecithin:cholesterol acyltransferase (LCAT) is the enzyme responsible for generation of the majority of the cholesteryl esters (CE) in human plasma. Although most plasma cholesterol esterification occurs on high-density lipoprotein (HDL), via alpha-LCAT activity, esterification also occurs on low-density lipoprotein (LDL) via the beta-activity of the enzyme. Computer threading techniques have provided a three-dimensional model for use in the structure-function analysis of the core and catalytic site of the LCAT protein, but the model does not extend to the N-terminal region of the enzyme, which may mediate LCAT interaction with lipoprotein substrates. In the present study, we have examined the functional consequences of deletion of the highly conserved hydrophobic N-terminal amino acids (residues 1-5) of human LCAT. Western blot analysis showed that the mutant proteins (Delta 1-Delta 5) were synthesized and secreted from transfected COS-7 cells at levels approximately equivalent to those of wild-type hLCAT. The secreted proteins had apparent molecular weights of 67 kDa, indicating that they were correctly processed and glycosylated during cellular transit. However, deletion of the first residue of the mature LCAT protein (Delta 1 mutant) resulted in a dramatic loss of alpha-LCAT activity (5% of wild type using reconstituted HDL substrate, rHDL), although this mutant retained full beta-LCAT activity (108% of wild-type using human LDL substrate). Removal of residues 1 and 2 (Delta 2 mutant) abolished alpha-LCAT activity and reduced beta-LCAT activity to 12% of wild type. Nevertheless, LCAT Delta 1 and Delta 2 mutants retained their ability to bind to rHDL and LDL lipoprotein substrates. The dramatic loss of enzyme activity suggests that the N-terminal residues of LCAT may be involved in maintaining the conformation of the lid domain and influence activation by the alpha-LCAT cofactor apoA-I (in Delta 1) and/or loss of enzyme activity (in Delta 1-Delta 5). Since the Delta 1 and Delta 2 mutants retain their ability to bind substrate, other factor(s), such as decreased access to the substrate binding pocket, may be responsible for the loss of enzyme activity.     

The role of Mg2+ and specific amino acid residues in the catalytic reaction of the major human abasic endonuclease: new insights from EDTA-resistant incision of acyclic abasic site analogs and site-directed mutagenesis.

Ape1, the major protein responsible for excising apurinic/apyrimidinic (AP) sites from DNA, cleaves 5' to natural AP sites via a hydrolytic reaction involving Mg2+. We report here that while Ape1 incision of the AP site analog tetrahydrofuran (F-DNA) was approximately 7300-fold reduced in 4 mM EDTA relative to Mg2+, cleavage of ethane (E-DNA) and propane (P-DNA) acyclic abasic site analogs was only 20 and 30-fold lower, respectively, in EDTA compared to Mg2+. This finding suggests that the primary role of the metal ion is to promote a conformational change in the ring-containing abasic DNA, priming it for enzyme-mediated hydrolysis. Mutating the proposed metal-coordinating residue E96 to A or Q resulted in a approximately 600-fold reduced incision activity for both P and F-DNA in Mg2+compared to wild-type. These mutants, while retaining full binding activity for acyclic P-DNA, were unable to incise this substrate in EDTA, pointing to an alternative or an additional function for E96 besides Mg2+-coordination. Other residues proposed to be involved in metal coordination were mutated (D70A, D70R, D308A and D308S), but displayed a relatively minor loss of incision activity for F and P-DNA in Mg2+, indicating a non-essential function for these amino acid residues. Mutations at Y171 resulted in a 5000-fold reduced incision activity. A Y171H mutant was fourfold less active than a Y171F mutant, providing evidence that Y171 does not operate as the proton donor in catalysis and that the additional role of E96 may be in establishing the appropriate active site environment via a hydrogen-bonding network involving Y171. D210A and D210N mutant proteins exhibited a approximately 25,000-fold reduced incision activity, indicating a critical role for this residue in the catalytic reaction. A D210H mutant was 15 to 20-fold more active than the mutants D210A or D210N, establishing that D210 likely operates as the leaving group proton donor.     

A second-site mutation at phenylalanine-137 that increases catalytic efficiency in the mutant aspartate-27----serine Escherichia coli dihydrofolate reductase

The adaptability of Escherichia coli dihydrofolate reductase (DHFR) is being explored by identifying second-site mutations that can partially suppress the deleterious effect associated with removal of the active-site proton donor aspartic acid-27. The Asp27----serine mutant DHFR (D27S) was previously characterized and the catalytic activity found to be greatly decreased at pH 7.0 [Howell et al. (1986) Science 231, 1123-1128]. Using resistance to trimethoprim (a DHFR inhibitor) in a genetic selection procedure, we have isolated a double-mutant DHFR gene containing Asp27----Ser and Phe137----Ser mutations (D27S+F137S). The presence of the F137S mutation increases kcat approximately 3-fold and decreases Km(DHF) approximately 2-fold over D27S DHFR values. The overall effect on kcat/Km(DHF) is a 7-fold increase. The D27S+F137S double-mutant DHFR is still 500-fold less active than wild-type DHFR at pH 7. Surprisingly, Phe137 is approximately 15 A from residue 27 in the active site and is part of a beta-bulge. We propose the F137S mutation likely causes its catalytic effect by slightly altering the conformation of D27S DHFR. This supposition is supported by the observation that the F137S mutation does not have the same kinetic effect when introduced into the wild-type and D27S DHFRs, by the altered distribution of two conformers of free enzyme [see Dunn et al. (1990)] and by a preliminary difference Fourier map comparing the D27S and D27S+F137S DHFR crystal structures.     

Homology modeling of the insect chitinase catalytic domain--oligosaccharide complex and the role of a putative active site tryptophan in catalysis

Knowledge-based protein modeling and substrate docking experiments as well as structural and sequence comparisons were performed to identify potential active-site residues in chitinase, a molting enzyme from the tobacco hornworm, Munduca sexta. We report here the identification of an active-site amino acid residue, W145. Several mutated forms of the gene encoding this protein were generated by site-directed mutagenesis, expressed in a baculovirus-insect cell-line system, and the corresponding mutant proteins were purified and characterized for their catalytic and substrate-binding properties. W145, which is present in the presumptive catalytic site, was selected for mutation to phenylalanine (F) and glycine (G), and the resulting mutant enzymes were characterized to evaluate the mechanistic role of this residue. The wild-type and W145F mutant proteins exhibited similar hydrolytic activities towards a tri-GlcNAc oligosaccharide substrate, but the former was approximately twofold more active towards a polymeric chitin-modified substrate. The W145G mutant protein was inactive towards both substrates, although it still retained its ability to bind chitin. Therefore, W145 is required for optimal catalytic activity but is not essential for binding to chitin. Measurement of kinetic constants of the wild-type and mutant proteins suggests that W145 increases the affinity of the enzyme for the polymeric substrate and also extends the alkaline pH range in which the enzyme is active.     

The role of subsite +2 of the Trichoderma reesei β-mannanase TrMan5A in hydrolysis and transglycosylation

The N-terminal catalytic module of β-mannanase TrMan5A from the filamentous fungus Trichoderma reesei is classified into family 5 of glycoside hydrolases. It is further classified in clan A with a (β/α)₈ barrel configuration and has two catalytic glutamates (E169 and E276). It has at least five other residues conserved in family 5. Sequence alignment revealed that an arginine (R171 in TrMan5A) is semi-conserved among β-mannanases in family 5. In a previously published mannobiose complex structure, this residue is positioned in hydrogen bonding distance from the C2 hydroxyl group of the mannose residue bound at the +2 subsite. To study the function of R171, mutants of this residue were constructed. The results show that arginine 171 is important for substrate binding and transglycosylation. A mutant of TrMan5A with the substitution R171K displayed retained activity on polymeric galactomannan but reduced activity on oligosaccharides due to an increase of K_m. While the wild-type enzyme produces mannobiose as dominant product from mannotetraose the R171K mutant shows an altered product profile, producing mannotriose and mannose. The cleavage pattern of mannotetraose was analysed with a method using isotope labelled water (H₂¹⁸O) and mass spectrometry which showed that the preferred productive binding mode of mannotetraose was shifted from subsite ?2 to +2 in the wild-type to subsite ?3 to +1 in the R171K mutant. Significant differences in product formation after manno-oligosaccharide incubation showed that the wild-type enzyme can perform transglycosylation on to saccharide acceptors while the R171K mutant cannot, likely due to loss of acceptor affinity. Interestingly, both enzymes show the ability to perform alcoholysis reactions with methanol and butanol, forming new β-linked glyco-conjugates. Furthermore, it appears that the wild-type enzyme produces mainly mannobiose conjugates using M₄ as substrate, while in contrast the R171K mutant produces mainly mannotriose conjugates, due to the altered subsite binding.     

Site-directed mutagenesis of Klebsiella aerogenes urease: identification of histidine residues that appear to function in nickel ligation, substrate binding, and catalysis.

Comparison of six urease sequences revealed the presence of 10 conserved histidine residues (H96 in the gamma subunit, H39 and H41 in beta, and H134, H136, H219, H246, H312, H320, and H321 in the alpha subunit of the Klebsiella aerogenes enzyme). Each of these residues in K. aerogenes urease was substituted with alanine by site-directed mutagenesis, and the mutant proteins were purified and characterized in order to identify essential histidine residues and assign their roles. The gamma H96A, beta H39A, beta H41A, alpha H312A, and alpha H321A mutant proteins possess activities and nickel contents similar to wild-type enzyme, suggesting that these residues are not essential for substrate binding, catalysis, or metal binding. In contrast, the alpha H134A, alpha H136A, and alpha H246A proteins exhibit no detectable activity and possess 53%, 6%, and 21% of the nickel content of wild-type enzyme. These results are consistent with alpha H134, alpha H136, and alpha H246 functioning as nickel ligands. The alpha H219A protein is active and has nickel (approximately 1.9% and approximately 80%, respectively, when compared to wild-type protein) but exhibits a very high Km value (1,100 +/- 40 mM compared to 2.3 +/- 0.2 mM for the wild-type enzyme). These results are compatible with alpha H219 having some role in facilitating substrate binding. Finally, the alpha H320A protein (Km = 8.3 +/- 0.2 mM) only displays approximately 0.003% of the wild-type enzyme activity, despite having a normal nickel content.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic activity of poliovirus RNA polymerases with mutations at the tyrosine residue of the conserved YGDD motif: isolation and characterization of polioviruses containing RNA polymerases with FGDD and MGDD sequences.

The poliovirus RNA-dependent RNA polymerase (3Dpol) shares a region of homology with all RNA polymerases, centered around the amino acid motif YGDD, which has been postulated to be involved in the catalytic activity of the enzyme. Using oligonucleotide site-directed mutagenesis, we substituted the tyrosine at this motif of the poliovirus RNA-dependent RNA polymerase with cysteine, histidine, isoleucine, methionine, phenylalanine, or serine. The enzymes were expressed in Escherichia coli, and in vitro enzyme activity was tested. The phenylalanine and methionine substitutions resulted in enzymes with activity equal to that of the wild-type enzyme. The cysteine substitution resulted in an enzyme with approximately 50% of the wild-type activity, while the serine substitution resulted in an enzyme with approximately 10% of the wild-type activity; the isoleucine and histidine substitutions resulted in background levels of enzyme activity. To assess the effects of the mutants in viral replication, the mutant polymerase genes were subcloned into the infectious cDNA clone of poliovirus. Transfection of poliovirus cDNA containing the phenylalanine mutation in 3Dpol gave rise to virus in all of the transfection trials, while cDNA containing the methionine mutation resulted in virus in only 3 of 40 transfections. Transfection of cDNAs containing the other substitutions at the tyrosine residue did not result in infectious virus. The recovered viruses demonstrated kinetics of replication similar to those of the wild-type virus, as measured by [3H]uridine incorporation at either 37 or 39 degrees C. RNA sequence analysis of the 3Dpol gene of both viruses demonstrated that the tyrosine-to-phenylalanine or tyrosine-to-methionine mutation was still present. No other differences in the 3Dpol gene between the wild-type and phenylalanine-containing virus were found. The virus containing the methionine mutation also contained two other nucleotide changes from the wild-type 3Dpol sequence; one resulted in a glutamic acid-to-aspartic acid change at amino acid 108 of the polymerase, and the other resulted in a C-to-T base change at nucleotide 6724, which did not result in an amino acid change. To confirm that the second amino acid mutation found in the 3Dpol gene of the methionine-substituted virus allowed for replication ability, a mutation corresponding to the glutamic acid-to-aspartic acid change was made in the polymerase containing the methionine substitution, and this double-mutant polymerase was expressed in E. coli. The double-mutant enzyme was as active as the wild-type enzyme under in vitro assay conditions.(ABSTRACT TRUNCATED AT 400 WORDS)     

Yeast mitochondrial DNA mutators with deficient proofreading exonucleolytic activity.

The MIP1 gene which encodes yeast mitochondrial DNA polymerase possesses in its N-terminal region the three motifs (Exo1, Exo2 and Exo3) which characterize the 3'-5' exonucleolytic domain of many DNA polymerases. By site directed mutagenesis we have substituted alanine or glycine residues for conserved aspartate residues in each consensus sequence. Yeast mutants were therefore generated that are capable of replicating mitochondrial DNA (mtDNA) and exhibit a mutator phenotype, as estimated by the several hundred-fold increase in the frequency of spontaneous mitochondrial erythromycin resistant mutants. By overexpressing the mtDNA polymerase from the GAL1 promoter as a major 140 kDa polypeptide, we showed that the wild-type enzyme possesses a mismatch-specific 3'-5' exonuclease activity. This activity was decreased by approximately 500-fold in the mutant D347A; in contrast, the extent of DNA synthesis was only slightly decreased. The wild-type mtDNA polymerase efficiently catalyses elongation of singly-primed M13 DNA to the full-length product. However, the mutant preferentially accumulates low molecular weight products. These data were extended to the two other mutators D171G and D230A. Glycine substitution for the Cys344 residue which is present in the Exo3 site of several polymerases generates a mutant with a slightly higher mtDNA mutation rate and a slightly lower 3'-5' exonucleolytic activity. We conclude that proofreading is an important determinant of accuracy in the replication of yeast mtDNA.     

Mucopolysaccharidosis type IIIB: characterisation and expression of wild-type and mutant recombinant alpha-N-acetylglucosaminidase and relationship with sanfilippo phenotype in an attenuated patient

Mucopolysaccharidosis type IIIB (MPS-IIB) is a lysosomal storage disorder characterised by the defective degradation of heparan sulfate due to a deficiency of alpha-N-acetylglucosaminidase (NAG). The clinical severity of MPS-IIIB ranges from an attenuated to severely affected Sanfilippo phenotype. This paper describes the expression and characterisation of wild-type recombinant NAG and the molecular characterisation of a previously identified R297X/F48L compound heterozygous MPS-IIIB patient with attenuated Sanfilippo syndrome. We have previously shown R297X to be the most common mutation in a cohort of Dutch and Australian patients, occurring at a frequency of approximately 12.5%. To date F48L has only been described in the proband. To determine the contribution of each mutation to the overall clinical phenotype of the patient, both mutant alleles were engineered into the wild-type NAG cDNA and expressed in Chinese hamster ovary cells. The wild-type NAG and F48L mutant alleles were also retrovirally expressed in MPS-IIIB skin fibroblasts. Residual NAG activity and the stability and maturation of immunoprecipitated NAG were determined for wild-type NAG and mutant NAG. The combined biochemical phenotypes of the two NAG mutant alleles demonstrated a good correspondence with the observed attenuated Sanfilippo phenotype of the patient.     

Switching kinetic mechanism and putative proton donor by directed mutagenesis of glutathione reductase

By directed mutagenesis of the cloned Escherichia coli gor gene encoding the flavoprotein glutathione reductase, Tyr-177 (the residue corresponding to Tyr-197 in the NADPH-binding pocket of the homologous human enzyme) was changed to phenylalanine (Y177F), serine (Y177S), and glycine (Y177G). The catalytic activity of the Y177F mutant was very similar to that of the wild-type enzyme, but that of the Y177S and Y177G mutants was substantially diminished. However, all three mutants retained the ability to protect the reduced flavin from adventitious oxidation, indicating that Tyr-177 does not act as a simple "lid" on the NADPH-binding pocket and that the protection of the reduced enzyme must be due largely to burial of the isoalloxazine ring in the protein. The wild-type enzyme and Y177F mutant displayed ping-pong kinetics, but the Y177S and Y177G mutants appeared to have switched to an ordered sequential mechanism. This could be explained by supposing that the enzyme normally functions by a hybrid kinetic mechanism and that the Y177S and Y177G mutations diverted flux from the ping-pong loop favored by the wild-type enzyme to an ordered sequential loop. The necessary change in the partitioning of the common E-NADPH intermediate could be caused by a slowing of the formation of the EH2 intermediate on the ping-pong loop, or by the observed concomitant fall in the Km for glutathione favoring flux through the ordered sequential loop. In another experiment, His-439, thought to act as a proton donor/acceptor in the glutathione-binding pocket, was mutated to a glutamine residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mutation of a critical arginine in the GTP-binding site of transglutaminase 2 disinhibits intracellular cross-linking activity

Transglutaminase type 2 (TG2; also known as G(h)) is a multifunctional protein involved in diverse cellular processes. It has two well characterized enzyme activities: receptor-stimulated signaling that requires GTP binding and calcium-activated transamidation or cross-linking that is inhibited by GTP. In addition to the GDP binding residues identified from the human TG2 crystal structure (Liu, S., Cerione, R. A., and Clardy, J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 2743-2747), we have previously implicated Ser171 in GTP binding, as binding is lost with glutamate substitution (Iismaa, S. E., Wu, M.-J., Nanda, N., Church, W. B., and Graham, R. M. (2000) J. Biol. Chem. 275, 18259-18265). Here, we have shown that alanine substitution of homologous residues in rat TG2 (Phe174 in the core domain or Arg476, Arg478, or Arg579 in barrel 1) does not affect TG activity but reduces or abolishes GTP binding and GTPgammaS inhibition of TG activity in vitro, indicating that these residues are important in GTP binding. Alanine substitution of Ser171 does not impair GTP binding, indicating this residue does not interact directly with GTP. Arg579 is particularly important for GTP binding, as isothermal titration calorimetry demonstrated a 100-fold reduction in GTP binding affinity by the R579A mutant. Unlike wild-type TG2 or its S171E or F174A mutants, which are sensitive to both trypsin and mu-calpain digestion, R579A is inherently more resistant to mu-calpain, but not trypsin, digestion, indicating reduced accessibility and/or flexibility of this mutant in the region of the calpain cleavage site(s). Basal TG activity of intact R579A stable SH-SY5Y neuroblastoma cell transfectants was slightly increased relative to wild-type transfectants and, in contrast to the TG activity of the latter, was further stimulated by muscarinic receptor-activated calcium mobilization. Thus, loss of GTP binding sensitizes TG2 to intracellular calcium concentrations. These findings are consistent with the notion that intracellularly, under physiological conditions, TG2 is maintained largely as a latent enzyme, its calcium-activated cross-linking activity being suppressed allosterically by guanine nucleotide binding.     

嗜甲基菌DM11菌株二氯甲烷脱卤素酶基因的定点诱变

为了研究嗜甲基菌（Methylophilus）DM11菌株二氯甲烷脱卤素酶的不同氨基酸残基在底物结合、谷胱甘肽（GSH）亲和以及催化活力中的作用,对编码该酶的基因进行了定点诱变研究。将保守的103位色氨酸（W）分别用苯丙氨酸（F）、缬氨酸（V）或天冬酞胺（N）替换,109位精氨酸（R）用亮氨酸（L）替换,117位色氨酸用酪氨酸（Y）或苯丙氨酸替换,得到6种突变酶。其中3种突变酶具有较低的活力,另外3种突变酶没有活力。突变酶W117Y的性质与野生型酶明显不同。