Crystal structures of two bacterial 3-hydroxy-3-methylglutaryl-CoA lyases suggest a common catalytic mechanism among a family of TIM barrel metalloenzymes cleaving carbon-carbon bonds

The enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the terminal steps in ketone body generation and leucine degradation. Mutations in this enzyme cause a human autosomal recessive disorder called primary metabolic aciduria, which typically kills victims because of an inability to tolerate hypoglycemia. Here we present crystal structures of the HMG-CoA lyases from Bacillus subtilis and Brucella melitensis at 2.7 and 2.3 A resolution, respectively. These enzymes share greater than 45% sequence identity with the human orthologue. Although the enzyme has the anticipated triose-phosphate isomerase (TIM) barrel fold, the catalytic center contains a divalent cation-binding site formed by a cluster of invariant residues that cap the core of the barrel, contrary to the predictions of homology models. Surprisingly, the residues forming this cation-binding site and most of their interaction partners are shared with three other TIM barrel enzymes that catalyze diverse carbon-carbon bond cleavage reactions believed to proceed through enolate intermediates (4-hydroxy-2-ketovalerate aldolase, 2-isopropylmalate synthase, and transcarboxylase 5S). We propose the name "DRE-TIM metallolyases" for this newly identified enzyme family likely to employ a common catalytic reaction mechanism involving an invariant Asp-Arg-Glu (DRE) triplet. The Asp ligates the divalent cation, while the Arg probably stabilizes charge accumulation in the enolate intermediate, and the Glu maintains the precise structural alignment of the Asp and Arg. We propose a detailed model for the catalytic reaction mechanism of HMG-CoA lyase based on the examination of previously reported product complexes of other DRE-TIM metallolyases and induced fit substrate docking studies conducted using the crystal structure of human HMG-CoA lyase (reported in the accompanying paper by Fu, et al. (2006) J. Biol. Chem. 281, 7526-7532). Our model is consistent with extensive mutagenesis results and can guide subsequent studies directed at definitive experimental elucidation of this enzyme's reaction mechanism.     

Crystal structure of human 3-hydroxy-3-methylglutaryl-CoA Lyase: insights into catalysis and the molecular basis for hydroxymethylglutaric aciduria

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase is a key enzyme in the ketogenic pathway that supplies metabolic fuel to extrahepatic tissues. Enzyme deficiency may be due to a variety of human mutations and can be fatal. Diminished activity has been explained based on analyses of recombinant human mutant proteins or, more recently, in the context of structural models for the enzyme. We report the experimental determination of a crystal structure at 2.1 A resolution of the recombinant human mitochondrial HMG-CoA lyase containing a bound activator cation and the dicarboxylic acid 3-hydroxyglutarate. The enzyme adopts a (betaalpha)(8) barrel fold, and the N-terminal barrel end is occluded. The structure of a physiologically relevant dimer suggests that substrate access to the active site involves binding across the cavity located at the C-terminal end of the barrel. An alternative hypothesis that involves substrate insertion through a pore proposed to extend through the barrel is not compatible with the observed structure. The activator cation ligands included Asn(275), Asp(42),His(233), and His(235); the latter three residues had been implicated previously as contributing to metal binding or enzyme activity. Arg(41), previously shown to have a major effect on catalytic efficiency, is also located at the active site. In the observed structure, this residue interacts with a carboxyl group of 3-hydroxyglutarate, the hydrolysis product of the competitive inhibitor 3-hydroxyglutaryl-CoA required for crystallization of human enzyme. The structure provides a rationale for the decrease in enzyme activity due to clinical mutations, including H233R, R41Q, D42H, and D204N, that compromise active site function or enzyme stability.     

Investigation of conserved acidic residues in 3-hydroxy-3-methylglutaryl-CoA lyase: implications for human disease and for functional roles in a family of related proteins

3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to form acetyl-CoA and acetoacetate. In metal-dependent aldol and Claisen reactions, acidic residues often function either as cation ligands or as participants in general acid/base catalysis. Site-directed mutagenesis was used to produce conservative substitutions for the conserved acidic residues Glu-37, Asp-42, Glu-72, Asp-204, Glu-279, and Asp-280. HMG-CoA lyase deficiency results from a human mutation that substitutes lysine for glutamate 279. The E279K mutation has also been engineered; expression in Escherichia coli produces an unstable protein. Substitution of alanine for glutamate 279 produces a protein that is sufficiently stable for isolation and retains substantial catalytic activity. However, thermal inactivation experiments demonstrate that E279A is much less stable than wild-type enzyme. HMG-CoA lyase deficiency also results from mutations at aspartate 42. Substitutions that eliminate a carboxyl group at residue 42 perturb cation binding and substantially lower catalytic efficiency (104-105-fold decreases in specific activity for D42A, D42G, or D42H versus wild-type). Substitutions of alanine for the other conserved acidic residues indicate the importance of glutamate 72. E72A exhibits a 200-fold decrease in kcat and >103-fold decrease in kcat/Km. E72A is also characterized by inflation in the Km for activator cation (26-fold for Mg2+; >200-fold for Mn2+). Similar, but less pronounced, effects are measured for the D204A mutant. E72A and D204A mutant proteins both bind stoichiometric amounts of Mn2+, but D204A exhibits only a 2-fold inflation in KD for Mn2+, whereas E72A exhibits a 12-fold inflation in KD (23 microm) in comparison with wild-type enzyme (KD = 1.9 microm). Acidic residues corresponding to HMG-CoA lyase Asp-42 and Glu-72 are conserved in the HMG-CoA lyase protein family, which includes proteins that utilize acetyl-CoA in aldol condensations. These related reactions may require an activator cation that binds to the corresponding acidic residues in this protein family.     

X-ray structure of human acid-beta-glucosidase,the defective enzyme in Gaucher disease

Gaucher disease, the most common lysosomal storage disease, is caused by mutations in the gene that encodes acid-β-glucosidase (GlcCerase). Type 1 is characterized by hepatosplenomegaly, and types 2 and 3 by early or chronic onset of severe neurological symptoms. No clear correlation exists between the ~200 GlcCerase mutations and disease severity, although homozygosity for the common mutations N370S and L444P is associated with non- neuronopathic and neuronopathic disease, respectively. We report the X-ray structure of GlcCerase at 2.0 Å resolution. The catalytic domain consists of a (β/α)₈ TIM barrel, as expected for a member of the glucosidase hydrolase A clan. The distance between the catalytic residues E235 and E340 is consistent with a catalytic mechanism of retention. N370 is located on the longest α-helix (helix 7), which has several other mutations of residues that point into the TIM barrel. Helix 7 is at the interface between the TIM barrel and a separate immunoglobulin-like domain on which L444 is located, suggesting an important regulatory or structural role for this non-catalytic domain. The structure provides the possibility of engineering improved GlcCerase for enzyme-replacement therapy, and for designing structure-based drugs aimed at restoring the activity of defective GlcCerase.     

Evaluation of 3-hydroxy-3-methylglutaryl-coenzyme A lyase arginine-41 as a catalytic residue: use of acetyldithio-coenzyme A to monitor product enolization

3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) lyase catalyzes the divalent cation-dependent cleavage of HMG-CoA to produce acetyl-CoA and acetoacetate. Arginine-41 is an invariant residue in HMG-CoA lyases. Mutation of this residue (R41Q) correlates with human HMG-CoA lyase deficiency. To evaluate the functional importance of arginine-41, R41Q and R41M recombinant mutant human HMG-CoA lyase proteins have been constructed, expressed, and purified. These mutant proteins retain structural integrity based on Mn(2+) binding and affinity labeling stoichiometry. R41Q exhibits a 10(5)-fold decrease in V(max); R41M activity is >or=10-fold lower than the activity of R41Q. Acetyldithio-CoA, an analogue of the reaction product, acetyl-CoA, has been employed to test the function of arginine-41, as well as other residues (e.g., aspartate-42 and histidine-233) implicated in catalysis. Acetyldithio-CoA supports enzyme-catalyzed exchange of the methyl protons of the acetyl group with solvent; exchange is dependent on the presence of Mg(2+) and acetoacetate. In comparison with wild-type human enzyme, D42A and H233A mutant enzymes exhibit 4-fold and 10-fold decreases, respectively, in the proton exchange rate. In contrast, R41Q and R41M mutants do not catalyze any substantial enzyme-dependent proton exchange. These results suggest a role for arginine-41 in deprotonation or enolization of acetyldithio-CoA and implicate this residue in the HMG-CoA cleavage reaction chemistry that leads to acetyl-CoA product formation. Assignment of arginine-41 as an active site residue is also supported by a homology model for HMG-CoA lyase based on the structure of 4-hydroxy-2-ketovalerate aldolase. This model suggests the proximity of arginine-41 to other amino acids (aspartate-42, glutamate-72, histidine-235) implicated as active site residues based on their function as ligands to the activator cation.     

Succinylpurinemic autism: increased sensitivity of defective adenylosuccinate lyase towards 4-hydroxy-2-nonenal

We studied the effect of trans-4-hydroxy-2-nonenal on the wild-type human adenylosuccinate lyase and on the enzyme from a patient compound-heterozygous for two missense mutations (P75A/D397Y; McKusick 103050.0003/103050.0004). Both the enzymes were inhibited by 10-50 microM trans-4-hydroxy-2-nonenal in a concentration-dependent manner by means of a mixed-type co-operative mechanism. A significantly stronger inhibition was noticed in the presence of the defective enzyme. Nonanal and trans-2,3-nonenal inhibited the enzymes to a less extent and at about 10-times higher concentrations. Hydroxylamine reversed the inhibition by trans-4-hydroxy-2-nonenal, trans-2,3-nonenal or nonanal in the case of the wild-type enzyme, but it was ineffective to reverse the inhibition by trans-4-hydroxy-2-nonenal on the defective enzyme. Dithiothreitol slightly decreased the inhibition exerted by trans-4-hydroxy-2-nonenal on both the wild-type and the defective adenylosuccinate lyase, while it did not produce practically any change in the presence of trans-2,3-nonenal or nonanal.     

The influence of conserved aromatic residues in 3-hydroxy-3-methylglutaryl-CoA synthase

In order to evaluate the potential contribution of conserved aromatic residues to the hydrophobic active site of 3-hydroxy-3-methylglutaryl-CoA synthase, site-directed mutagenesis was employed to produce Y130L, Y163L, F204L, Y225L, Y346L, and Y376L proteins. Each mutant protein was expressed at levels comparable with wild-type enzyme and was isolated in highly purified form. Initial kinetic characterization indicated that F204L exhibits a substantial (>300-fold) decrease in catalytic rate (kcat). Upon modification with the mechanism-based inhibitor, 3-chloropropionyl-CoA, or in formation of a stable binary complex with acetoacetyl-CoA, F204L exhibits binding stoichiometries comparable with wild-type enzyme, suggesting substantial retention of active site integrity. Y130L and Y376L exhibit inflated values (80- and 40-fold, respectively) for the Km for acetyl-CoA in the acetyl-CoA hydrolysis partial reaction; these mutants also exhibit an order of magnitude decrease in kcat. Formation of the acetyl-S-enzyme reaction intermediate by Y130L, F204L, and Y376L proceeds slowly in comparison with wild-type enzyme. However, solvent exchange into the thioester carbonyl oxygen of these acetyl-S-enzyme intermediates is not slow in comparison with previous observations for D159A and D203A mutants, which also exhibit slow acetyl-S-enzyme formation. The magnitude of the differential isotope shift upon exchange of H218O into [13C]acetyl-S-enzyme suggests a polarization of the thioester carbonyl and a reduction in bond order. Such an effect may substantially contribute to the upfield 13C NMR shift observed for [13C]acetyl-S-enzyme. The influence on acetyl-S-enzyme formation, as well as observed kcat (F204L) and Km (Y130L; Y376L) effects, implicate these invariant residues as part of the catalytic site. Substitution of phenylalanine (Y130F, Y376F) instead of leucine at residues 130 and 376 diminishes the effects on catalytic rate and substrate affinity observed for Y130L and Y376L, underscoring the influence of aromatic side chains near the active site.     

Skipping of exon 2 and exons 2 plus 3 of HMG-CoA lyase (HL) gene produces the loss of beta sheets 1 and 2 in the recently proposed (beta-alpha)8 TIM barrel model of HL

HMG-CoA lyase (HL) deficiency is a rare autosomal recessive genetic disorder that affects ketogenesis and leucine catabolism. We report a new Spanish patient who bears the frequent nonsense mutation G109T (Mediterranean mutation). This mutation can produce aberrant splicing with three mRNA variants: one of the expected size, the second with deletion of exon 2, and the third with deletion of exons 2 and 3. Recently our group proposed a 3D model for human HL containing a (beta-alpha)(8) (TIM) barrel structure. We have studied the effect of the deletions of exon 2 and exons 2 plus 3 on the proposed HL model. Exon 2 skipping led to the loss of beta-sheet 1, and the skipping of exons 2 and 3 caused the disappearance of alpha helix 1 and beta-sheets 1 and 2.-     

3-Hydroxy-3-methylglutaryl coenzyme A lyase: affinity labeling of the Pseudomonas mevalonii enzyme and assignment of cysteine-237 to the active site.

Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) lyase is irreversibly inactivated by the reactive substrate analog 2-butynoyl-CoA. Enzyme inactivation, which follows pseudo-first-order kinetics, is saturable with a KI = 65 microM and a limiting k(inact) of 0.073 min-1 at 23 degrees C, pH 7.2. Protection against inactivation is afforded by the competitive inhibitor 3-hydroxyglutaryl-CoA. Labeling of the bacterial enzyme with [1-14C]-2-butynoyl-CoA demonstrates that inactivation coincides with covalent incorporation of inhibitor, with an observed stoichiometry of modification of 0.65 per site. Avian HMG-CoA lyase is also irreversibly inactivated by 2-butynoyl-CoA with a stoichiometry of modification of 0.9 per site. Incubation of 2-butynoyl-CoA with mercaptans such as dithiothreitol results in the formation of a UV absorbance peak at 310 nm. Enzyme inactivation is also accompanied by the development of a UV absorbance peak at 310 nm indicating that 2-butynoyl-CoA modifies a cysteine residue in HMG-CoA lyase. Tryptic digestion and reverse-phase HPLC of the affinity-labeled protein reveal a single radiolabeled peptide. Isolation and sequence analysis of this peptide and a smaller chymotryptic peptide indicate that the radiolabeled residue is contained within the sequence GGXPY. Mapping of this peptide within the cDNA-deduced sequence of P. mevalonii HMG-CoA lyase [Anderson, D. H., & Rodwell, V. W. (1989) J. Bacteriol. 171, 6468-6472] confirms that a cysteine at position 237 is the site of modification. These data represent the first identification of an active-site residue in HMG-CoA lyase.     

Identification of the catalytically important histidine of 3-hydroxy-3-methylglutaryl-coenzyme A reductase.

We identify His381 of Pseudomonas mevalonii 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase as the basic residue functional in catalysis. The catalytic domain of 20 HMG-CoA reductases contains a single conserved histidine (His381 of the P. mevalonii enzyme). Diethyl pyrocarbonate inactivated the P. mevalonii enzyme, and hydroxylamine partially restored activity. We changed His381 to alanine, lysine, asparagine, and glutamine. The mutant proteins were overexpressed, purified to homogeneity, and characterized. His381 mutant enzymes were not inactivated by diethyl pyrocarbonate. All four mutant enzymes exhibited wild-type crystal morphology and chromatographed on substrate affinity supports like wild-type enzyme. The mutant enzymes had low catalytic activity (Vmax 0.06-0.5% that of wild-type enzyme), but Km values approximated those for wild-type enzyme. For wild-type enzyme and mutant enzymes H381A, H381N, and H381Q, Km values at pH 8.1 were 0.45, 0.27, 3.7, and 0.71 mM [(R,S)-mevalonate]; 0.05, 0.03, 0.20, and 0.11 mM [coenzyme A]; 0.22, 0.14, 0.81, and 0.62 mM [NAD+]. Km values at pH 11 for wild-type enzyme and mutant enzyme H381K were 0.32 and 0.75 mM [(R,S)-mevalonate]; 0.24 and 0.50 mM [coenzyme A]; 0.15 and 1.23 mM [NAD+]. Both pK values for the enzyme-substrate complex increased relative to wild-type enzyme (by 1-2.5 pH units for pK1 and by 0.5-1.3 pH units for pK2). For mutant enzyme H381K, the pK1 of 10.2 is consistent with lysine acting as a general base at high pH. His381 of P. mevalonii HMG-CoA reductase, and consequently the histidine of the consensus Leu-Val-Lys-Ser-His-Met-Xaa-Xaa-Asn-Arg-Ser motif of the catalytic domain of eukaryotic HMG-CoA reductases, thus is the general base functional in catalysis.     

Structural basis for type I and type II deficiencies of antithrombotic plasma protein C: Patterns revealed by three-dimensional molecular modelling of mutations of the protease domain

Familial deficiency of protein C is associated with inherited thrombophilia. To explore how specific missense mutations might cause observed clinical phenotypes, known protein C missense mutations were mapped onto three-dimensional homology models of the protein C protease domain, and the implications for domain folding and structure were evaluated. Most Type I missense mutations either replaced internal hydrophobic residues (I201T, L223F, A259V, A267T, A346T, A346V, G376D) or nearby interacting residues (I403M, T298M, Q184H), thus disrupting the packing of internal hydrophobic side chains, or changed hydrophilic residues, thus disrupting ion pairs (N256D, R178W). Mutations (P168L, R169W) at the activation site destabilized the region containing the activation peptide structure. Most Type II mutations involved solvent-exposed residues and were clustered either in a positively charged region (R147W, R157Q, R229Q, R352W) or were located in or near the active site region (S252N, D359N, G381S, G391S, H211Q). The cluster of arginines 147, 157, 229, and 352 may identify a functionally important exosite. Identification of the spatial relationships of natural mutations in the protein C model is helpful for understanding manifestations of protein C deficiency and for identification of novel, functionally important molecular features and exosites. © 1994 John Wiley & Sons, Inc.     

Fabry disease: thirty-five mutations in the alpha-galactosidase A gene in patients with classic and variant phenotypes.

BACKGROUND: Fabry disease, an X-linked inborn error of glycosphingolipid catabolism, results from mutations in the alpha-galactosidase A (alpha-Gal A) gene located at Xq22.1. To determine the nature and frequency of the molecular lesions causing the classical and milder variant Fabry phenotypes and for precise carrier detection, the alpha-Gal A lesions in 42 unrelated Fabry hemizygotes were determined. MATERIALS AND METHODS: Genomic DNA was isolated from affected probands and their family members. The seven alpha-galactosidase A exons and flanking intronic sequences were PCR amplified and the nucleotide sequence was determined by solid-phase direct sequencing. RESULTS: Two patients with the mild cardiac phenotype had missense mutations, I9IT and F113L, respectively. In 38 classically affected patients, 33 new mutations were identified including 20 missense (MIT, A31V, H46R, Y86C, L89P, D92Y, C94Y, A97V, R100T, Y134S, G138R, A143T, S148R, G163V, D170V, C202Y, Y216D, N263S, W287C, and N298S), two nonsense (Q386X, W399X), one splice site mutation (IVS4 + 2T-->C), and eight small exonic insertions or deletions (304del1, 613del9, 777del1, 1057del2, 1074del2, 1077del1, 1212del3, and 1094ins1), which identified exon 7 as a region prone to gene rearrangements. In addition, two unique complex rearrangements consisting of contiguous small insertions and deletions were found in exons 1 and 2 causing L45R/H46S and L120X, respectively. CONCLUSIONS: These studies further define the heterogeneity of mutations causing Fabry disease, permit precise carrier identification and prenatal diagnosis in these families, and facilitate the identification of candidates for enzyme replacement therapy.     

Site-directed mutagenesis within the central constriction site of ScrY (sucroseporin): effect on ion transport and comparison of maltooligosaccharide binding to LamB of Escherichia coli

The 3-D structures of the maltooligosaccharide-specific LamB-channel of Escherichia coli (also called maltoporin) and sucrose-specific ScrY (sucroseporin) are known from X-ray crystallography. The central constriction of the channels formed by the external loop 3 is controlled by a number of different amino acids. The most prominent one of these, N192, D201 and F204, were replaced by site-directed mutagenesis into those of LamB, which, according to the 3-D model of both channels are localized at similar places. The ScrY single mutants ScrYN192R, ScrYD201Y and ScrYF204D and the ScrY triple mutant ScrY3113 (N192R + D201Y + F204D) were created together with the triple mutant ScrY3213, which lacks also amino acids 1 to 61 from the N-terminal end. The mutant proteins were purified to homogeneity and were reconstituted into lipid bilayer membranes. In these experiments, the single-channel conductance of the mutants in different salt solutions and the stability constants for binding of different maltooligosaccharides to the mutant channels was measured using titration experiments with carbohydrates. The carbohydrate-induced block of the channel function could also be used for the study of current noise through the different mutant ScrY-channels. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k1 and k-1) of carbohydrate-binding to the binding site inside the channels. The results suggest that both on- and off-rate constants were affected by the mutations. Most of them showed a substantial effect on carbohydrate binding kinetics. Nevertheless, single-channel conductance and carbohydrate binding of ScrY3113 mutant were still different from that of LamB, suggesting that not only the amino acids of the central constriction but also the general architecture of both channels have a substantial influence on channel properties.     

Prediction of substrate-binding site and elucidation of catalytic residue of a phytase from Bacillus sp

The present study primarily deals with the identification of substrate-binding site and elucidation of catalytic residue of the phytase from Bacillus sp. (Genbank Accession No. EF536824) employing molecular modeling and site-directed mutagenesis. Homology-based modeling of the Bacillus phytase revealed β-propeller structure with twelve active-site aminoacid residues, viz., D75, R77, Y78, H138, Q140, D189, D190, E191, Y238, Y239, N346 and R348. Docking of substrate Ins(1,2,3,4,5,6)hexakisphosphate with the phytase model disclosed interaction of Y78 residue with the sixth position phosphate, while D75 and R77 residues revealed hydrogen bonding with the fifth position phosphate of the phytate. Analysis of hydrolysis products of phytate indicated the sequential removal of alternate phosphates, resulting in the formation of final product Ins triphosphate. Mutant phytases Y78A/F, derived from site-directed mutagenesis, exhibited complete loss of enzyme activity despite substrate binding, thereby suggesting the intrinsic role of Y78 residue in the catalytic activity. The Bacillus mutant phytases can be used to generate enzyme crystals complexed with phytate and lower Ins phosphates for indepth analysis of substrate binding and catalytic activity of the enzyme.     

HMG CoA lyase deficiency: identification of five causal point mutations in codons 41 and 42, including a frequent Saudi Arabian mutation, R41Q.

The hereditary deficiency of 3-hydroxy-3-methylglutaryl (HMG) CoA lyase (HL; OMIM 246450 [http://www3.ncbi.nlm.nih. gov:80/htbin-post/Omim/dispmim?246450]) results in episodes of hypoketotic hypoglycemia and coma and is reported to be frequent and clinically severe in Saudi Arabia. We found genetic diversity among nine Saudi HL-deficient probands: six were homozygous for the missense mutation R41Q, and two were homozygous for the frameshift mutation F305fs(-2). In 32 non-Saudi HL-deficient probands, we found three R41Q alleles and also discovered four other deleterious point mutations in codons 41 and 42: R41X, D42E, D42G, and D42H. In purified mutant recombinant HL, all four missense mutations in codons 41 and 42 cause a marked decrease in HL activity. We developed a screening procedure for HL missense mutations that yields residual activity at levels comparable to those obtained using purified HL peptides. Codons 41 and 42 are important for normal HL catalysis and account for a disproportionate 21 (26%) of 82 of mutant alleles in our group of HL-deficient probands.     

Residues Y179 and H101 of a hydrophobic patch of factor VII are involved in activation by factor Xa

We studied factor Xa activation of human factor VII in hopes of identifying factor VII residues, not adjacent to the cleavage site, involved in this interaction. We made eight factor VIIs with single mutations (N100A, H101A, D102Q, L144A, R147A, Y179A, D186A, and F256A) and two factor VIIs with multiple mutations [MM3 (L144A/R147A/D186A) and MM4 (N100A/H101A/Y179A/F256A)]. Residues in MM3 have previously been identified as affecting factor X activation, and the residues of MM4 are located at a hydrophobic patch of factor VII on the opposite side of the catalytic domain from those in MM3. Only H101A, Y179A, and MM4 were activated significantly more slowly than the wild type. Results of our kinetic analyses showed that the catalytic efficiency of factor Xa for activation of factor VII was 176- and 234-fold higher than that for H101A andY179A, respectively. All the mutants with measurable activity had affinities for tissue factor similar to those of the wild type. The activated hydrophobic patch residues, except N100A, which is adjacent to one of the catalytic residues, had normal activities toward both a small peptide substrate and factor X. The rest of the activated mutants (except D102Q with no activity) had reduced activities toward the small substrate (except R147A) and factor X. We conclude that factor VII activation by factor Xa and factor VIIa's catalytic interaction with factor X involve different regions in the catalytic domain, and residues H101 and Y179, part of an aromatic hydrophobic patch, are specifically involved in factor Xa activation of factor VII.     

Analysis of essential amino acid residues for catalytic activity of cis-epoxysuccinate hydrolase from Bordetella sp. BK-52

cis-Epoxysuccinate hydrolase (CESH) from Bordetella sp. BK-52, an epoxide hydrolase (EH), catalyzes the stereospecific hydrolysis of cis-epoxysuccinate to d(?)-tartrate. The enzyme, which shows no homology to other reported EHs, belongs to the DUF849 superfamily of prokaryotic proteins, which have unknown function. Metal composition analysis revealed that the CESH is a Zn²⁺-dependent enzyme with an approximately 1:1 molar ratio of zinc to enzyme. The results of an ¹⁸O-labeling study suggest that the enzyme catalyzes epoxide hydrolysis by means of a one-step mechanism. We evaluated the relationship between the structure and function of the enzyme by means of sequence alignment, modeling, substrate binding, and reaction kinetics studies. The CESH has a canonical (β/α)₈ TIM barrel fold, and we used site-directed mutagenesis to identify eight residues (H47, H49, R51, T82, Y138, N140, W164, and D251) as being localized to the active site or highly conserved. On the basis of these results and theoretical considerations, we identified H47 and H49 as zinc-binding ligands, and we propose that a zinc atom and R51, T82, Y138, N140, W164, and D251 are the catalytic residues and participate in substrate binding. In summary, the structure and catalytic mechanism of the CESH from Bordetella sp. BK-52 differ from those of classic EHs, which have an α/β hydrolase fold, act via a two-step catalytic mechanism, and do not require cofactors, prosthetic groups, or metal ions.     

Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia

Heterozygous glucokinase (GCK) mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY). Over 600 GCK mutations have been reported of which ～65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for <10% of mutations. The aim of this study was to determine the minimum prevalence of GCK-MODY amongst diabetic subjects in Slovakia by sequencing GCK in 100 Slovakian probands with a phenotype consistent with GCK-MODY and to explore the pathogenicity of identified variants through family and functional studies. Twenty-two mutations were identified in 36 families (17 missense) of which 7 (I110N, V200A, N204D, G258R, F419S, c.580-2A>C, c.1113-1114delGC) were novel. Parental DNA was available for 22 probands (covering 14/22 mutations) and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N) mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI) between 0.6 to <0.001 compared to wild-type GCK (1.0). For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP) -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50) 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]). Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]). The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.     

A computational method to characterize the missense mutations in the catalytic domain of GAA protein causing Pompe disease

Pompe disease is an autosomal recessive lysosomal storage disease caused by acid α-glucosidase (GAA) deficiency, resulting in intralysosomal accumulation of glycogen, including cardiac, skeletal, and smooth muscle cells. The GAA gene is located on chromosome 17 (17q25.3), the GAA protein consists of 952 amino acids; of which 378 amino acids (347-726) falls within the catalytic domain of the protein and comprises of active sites (518 and 521) and binding sites (404, 600, 616, and 674). In this study, we used several computational tools to classify the missense mutations in the catalytic domain of GAA for their pathogenicity and stability. Eight missense mutations (R437C, G478R, N573H, Y575S, G605D, V642D, L705P, and L712P) were predicted to be pathogenic and destabilizing to the protein structure. These mutations were further subjected to phenotyping analysis using SNPeffect 4.0 to predict the chaperone binding sites and structural stability of the protein. The mutations R437C and G478R were found to compromise the chaperone-binding activity with GAA. Molecular docking analysis revealed that the G478R mutation to be more significant and hinders binding to the DNJ (Miglustat) compared with the R437C. Further molecular dynamic analysis for the two mutations demonstrated that the G478R mutation was acquired higher deviation, fluctuation, and lower compactness with decreased intramolecular hydrogen bonds compared to the mutant R437C. These data are expected to serve as a platform for drug design against Pompe disease and will serve as an ultimate tool for variant classification and interpretations.     

3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli.

Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.