Determinants of enzymatic specificity in the Cys-Met-metabolism PLP-dependent enzymes family: crystal structure of cystathionine gamma-lyase from yeast and intrafamiliar structure comparison

The crystal structure of cystathionine gamma-lyase (CGL) from yeast has been solved by molecular replacement at a resolution of 2.6 A. The molecule consists of 393 amino acid residues and one PLP moiety and is arranged in the crystal as a tetramer with D2 symmetry as in other related enzymes of the Cys-Met-metabolism PLP-dependent family like cystathionine beta-lyase (CBL). A structure comparison with other family members revealed surprising insights into the tuning of enzymatic specificity between the different family members. CGLs from yeast or human are virtually identical at their active sites to cystathionine gamma-synthase (CGS) from E. coli. Both CGLs and bacterial CGSs exhibit gamma-synthase and gamma-lyase activities depending on their position in the metabolic pathway and the available substrates. This group of enzymes has a glutamate (E333 in yeast CGL) which binds to the distal group of cystathionine (CTT) or the amino group of cysteine. Plant CGSs use homoserine phosphate instead of O-succinyl-homoserine as one substrate. This is reflected by a partially different active site structure in plant CGSs. In CGL and CBL the pseudosymmetric substrate must dock at the active site in different orientations, with S in gamma-position (CBL) or in delta-position (CGL). The conserved glutamate steers the substrate as seen in other CGLs. In CBLs this position is occupied by either tyrosine or hydrophobic residues directing binding of CTT such that S is in the in gamma-position. In methionine gamma-lyase a hydrophic patch operates as recognition site for the methyl group of the methionine substrate.     

Solution NMR structure of CGL2373, a polyketide cyclase-like protein from Corynebacterium glutamicum

Protein CGL2373 from Corynebacterium glutamicum was previously proposed to be a member of the polyketide_cyc2 family, based on amino-acid sequence and secondary structure features derived from NMR chemical shift assignments. We report here the solution NMR structure of CGL2373, which contains three α-helices and one antiparallel β-sheet and adopts a helix-grip fold. This structure shows moderate similarities to the representative polyketide cyclases, TcmN, WhiE, and ZhuI. Nevertheless, unlike the structures of these homologs, CGL2373 structure looks like a half-open shell with a much larger pocket, and key residues in the representative polyketide cyclases for binding substrate and catalyzing aromatic ring formation are replaced with different residues in CGL2373. Also, the gene cluster where the CGL2373-encoding gene is located in C. glutamicum contains additional genes encoding nucleoside diphosphate kinase, folylpolyglutamate synthase, and valine-tRNA ligase, different from the typical gene cluster encoding polyketide cyclase in Streptomyces. Thus, although CGL2373 is structurally a polyketide cyclase-like protein, the function of CGL2373 may differ from the known polyketide cyclases and needs to be further investigated. The solution structure of CGL2373 lays a foundation for in silico ligand screening and binding site identifying in future functional study.     

Essential requirements for substrate binding affinity and selectivity toward human CYP2 family enzymes

A detailed analysis of substrate selectivity within the cytochrome P450 2 (CYP2) family is reported. From a consideration of specific interactions between drug substrates for human CYP2 family enzymes and the putative active sites of CYP2A6, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP2E1, it is likely that the number and disposition of hydrogen bond donor/acceptors and aromatic rings within the various P450 substrate molecules determines their enzyme selectivity and binding affinity, together with directing their preferred routes of metabolism by the CYP2 enzymes concerned. Although many aliphatic residues are present in most P450 active sites, it would appear that their main contribution centers around hydrophobic interactions and desolvation processes accompanying substrate binding. Molecular modeling studies based on the recent CYP2C5 crystal structure appear to show close agreement with site-directed mutagenesis experiments and with information on substrate metabolism and selectivity within the CYP2 family.     

Ligand interactions of the Coprinopsis cinerea galectins

The basidiomycete Coprinopsis cinerea (Coprinus cinereus) expresses two fruiting body-specific isolectins (CGL1 and CGL2) that belong to the family of galectins. Understanding the role of these beta-galactoside binding lectins is still in the beginning. Even though the prerequisites for substrate binding are well understood, it is not known how discrimination between potential substrates is achieved and what kind of influence this has on the function in a distinct cellular context. Precise knowledge of the expression of galectins and their ligands will aid in elucidating their function. In Coprinopsis, the developmentally regulated ligands for galectins co-localise with galectin expression in the veil surrounding the developing primordium and the outer cells of the young stipe. In addition, galectin ligands are observed in the hymenium. The subcellular localisation of the galectin ligands suggests these to be present in cellular compartments distinct from galectin transport. The sensitivity of the in situ interactions with exogenous galectin towards detergents and organic solvents infers that these ligands are lipid-borne. Accordingly, lipid fractions from primordia are shown to contain galectin-binding compounds. Based on these results and the determined binding specificity towards substituted beta-galactosides we hypothesise that beta-galactoside-containing lipids (basidiolipids) found in mushrooms are physiological ligands for the galectins in C. cinerea.     

Structural basis for chitotetraose coordination by CGL3, a novel galectin-related protein from Coprinopsis cinerea

Recent advances in genome sequencing efforts have revealed an abundance of novel putative lectins. Among these, many galectin-related proteins, characterized by many conserved residues but intriguingly lacking critical amino acids, have been found in all corners of the eukaryotic superkingdom. Here we present a structural and biochemical analysis of one representative, the galectin-related lectin CGL3 found in the inky cap mushroom Coprinopsis cinerea. This protein contains all but one conserved residues known to be involved in β-galactoside binding in galectins. A Trp residue strictly conserved among galectins is changed to an Arg in CGL3 (R81). Accordingly, the galectin-related protein is not able to bind lactose. Screening of a glycan array revealed that CGL3 displays preference for oligomers of β1-4-linked N-acetyl-glucosamines (chitooligosaccharides) and GalNAcβ1-4GlcNAc (LacdiNAc). Carbohydrate-binding affinity of this novel lectin was quantified using isothermal titration calorimetry, and its mode of chitooligosaccharide coordination not involving any aromatic amino acid residues was studied by X-ray crystallography. Structural information was used to alter the carbohydrate-binding specificity and substrate affinity of CGL3. The importance of residue R81 in determining the carbohydrate-binding specificity was demonstrated by replacing this Arg with a Trp residue (R81W). This single-amino-acid change led to a lectin that failed to bind chitooligosaccharides but gained lactose binding. Our results demonstrate that, similar to the legume lectin fold, the galectin fold represents a conserved structural framework upon which dramatically altered specificities can be grafted by few alterations in the binding site and that, in consequence, many metazoan galectin-related proteins may represent lectins with novel carbohydrate-binding specificities.     

Caenorhabditis elegans N-glycan Core β-galactoside Confers Sensitivity towards Nematotoxic Fungal Galectin CGL2

The physiological role of fungal galectins has remained elusive. Here, we show that feeding of a mushroom galectin, Coprinopsis cinerea CGL2, to Caenorhabditis elegans inhibited development and reproduction and ultimately resulted in killing of this nematode. The lack of toxicity of a carbohydrate-binding defective CGL2 variant and the resistance of a C. elegans mutant defective in GDP-fucose biosynthesis suggested that CGL2-mediated nematotoxicity depends on the interaction between the galectin and a fucose-containing glycoconjugate. A screen for CGL2-resistant worm mutants identified this glycoconjugate as a Galβ1,4Fucα1,6 modification of C. elegans N-glycan cores. Analysis of N-glycan structures in wild type and CGL2-resistant nematodes confirmed this finding and allowed the identification of a novel putative glycosyltransferase required for the biosynthesis of this glycoepitope. The X-ray crystal structure of a complex between CGL2 and the Galβ1,4Fucα1,6GlcNAc trisaccharide at 1.5 Å resolution revealed the biophysical basis for this interaction. Our results suggest that fungal galectins play a role in the defense of fungi against predators by binding to specific glycoconjugates of these organisms.     

Protein farnesyl transferase target selectivity is dependent upon peptide stimulated product release

Protein farnesyl transferase (FTase) catalyzes transfer of a 15 carbon farnesyl lipid to cysteine in the C-terminal Ca1a2X sequence of numerous proteins including Ras. Previous studies have shown that product release is rate limiting and is dependent on binding of either a new peptide or isoprenoid diphosphate substrate. While considerable progress has been made in understanding how FTase distinguishes between related target proteins, the relative importance of the two pathways for product release on substrate selectivity is unclear. A detailed analysis of substrate stimulated product release has now been performed and provides new insights into the mechanism of FTase target selectivity. To clarify how FTase selects between different Ca1a2X sequences, we have examined the competition of various peptide substrates for modification with the isoprenoids farnesyl diphosphate (FPP) and anilinogeranyl diphosphate (AGPP). We find that reactivity of some competing peptides is correlated with apparent Kmpeptide, while the reactivity of others is predicted by the selectivity factor apparent kcat/Kmpeptide. The peptide target selectivity also depends on the structure of the isoprenoid donor. Additionally, we observe two peptide substrate concentration dependent maxima and substrate inhibition in the steady-state reaction which require a minimum of three peptide binding states for the steady-state FTase reaction mechanism. We propose a model for the FTase reaction mechanism that, in addition to FPP stimulated product release, incorporates peptide binding to the FTase-FPP complex and the formation of an FTase-product-peptide complex followed by product release leading to an inhibitory FTase-peptide complex as a natural consequence of catalysis to explain these results.     

Divergent Metabolic Phenotype between Two Sisters with Congenital Generalized Lipodystrophy Due to Double AGPAT2 Homozygous Mutations. A Clinical,Genetic and In Silico Study

Congenital generalized lipodystrophy (CGL) is a rare autosomal recessive disorder characterized by extreme reduction of white adipose tissue (WAT) mass. CGL type 1 is the most frequent form and is caused by mutations in AGPAT2. Genetic and clinical studies were performed in two affected sisters of a Chilean family. These patients have notoriously dissimilar metabolic abnormalities that correlate with differential levels of circulating leptin and soluble leptin receptor fraction. Sequencing of AGPAT2 exons and exon-intron boundaries revealed two homozygous mutations in both sisters. Missense mutation c.299G>A changes a conserved serine in the acyltransferase NHX4D motif of AGPAT2 (p.Ser100Asn). Intronic c.493-1G>C mutation destroy a conserved splicing site that likely leads to exon 4 skipping and deletion of whole AGPAT2 substrate binding domain. In silico protein modeling provided insights of the mechanisms of lack of catalytic activity owing to both mutations.     

Tests of candidate genes in breed cross populations for QTL mapping in livestock

In recent years, several F₂ crosses between outbred lines of livestock have been developed to identify quantitative trait loci (QTL). These populations are valuable for further genetic analysis, including positional candidate gene loci (CGL). Analysis of CGL in F₂ populations is, however, hindered by extensive between-breed linkage disequilibrium (LD). The objectives here were to develop and evaluate three tests for CGL in simulated F₂ breed-cross populations. 1) A standard association test, based on the fixed effect of CGL genotype. This test was significant for CGL at considerable distances from the QTL. 2) A marker-assisted association test, based on a test at the CGL of the fixed effect of CGL genotype in a breed-cross QTL interval mapping model. This removed the impact of between-breed LD, but was not powerful in detecting CGL closely linked to the QTL, unless the CGL was the QTL. 3) An F-drop test, comparing F ratios for a QTL at the CGL with and without the CGL included as fixed effect. It had low power to distinguish close from distant CGL. Power to distinguish two CGL within 10 cM from the QTL was limited and little improved by including QTL effects associated with markers to remove between-breed LD, although the power was greater when one of the CGL was the causative mutation. Therefore, while we conclude that candidate gene tests in QTL mapping populations must be interpreted with caution, we now have a clearer picture of the value of candidate gene tests in these populations.     

Understanding the substrate selectivity and the product regioselectivity of Orf2-catalyzed aromatic prenylations

Orf2, a recently identified prenyltransferase of aromatic natural products, displays relaxed substrate selectivity and interesting product regioselectivity. This gives rise to the opportunity to engineer the active site to tune the functionality of terpenoids for therapeutic applications. The structural basis of substrate binding has been determined, but the source of the observed substrate selectivity and product regioselectivity cannot be completely understood on the basis of the static picture that the crystal structures of Orf2 and its complexes afford. The electron density and B-factors of the substrates, particularly those of 1,6-dihydroxynaphthalene, suggest significant conformational fluctuation in the Orf2 binding site. We thoroughly explored the binding of 1,6-dihydroxynaphthalene and quantitatively evaluated the relative free energies of three binding states that we identified in terms of a two-dimensional potential of mean force. The available experimental orientation, which gives the major prenylated product of 1,6-dihydroxynaphthalene, corresponds to the global free energy minimum. Two alternative binding states were identified on the calculated free energy surface, and both are readily accessible at 300 K. The alternative binding conformations were extracted from the potential of mean force calculation and were subjected to further validation against the experimental X-ray diffraction data using a refinement protocol supplemented with a hybrid quantum mechanical and molecular mechanical energy function. The agreement was excellent as indicated by the R and Rfree factors that were comparable to that obtained for the published orientation using a similar protocol. These binding states are the origin of the selectivity and regioselectivity in Orf2-catalyzed aromatic prenylations. Our analyses also suggest that Ser214 and Tyr288, forming hydrogen bonds with the alternative binding states of 1,6-dihydroxynaphthalene and flaviolin, are good candidates for site-directed mutagenesis, and changing them to, for example, their hydrophobic counterparts would affect the substrate selectivity and product regioselectivity.     

Oviductal Localization of the Cortical Granule Lectin Ligand Involved in the Block to Polyspermy of Xenopus Laevis

The fertilization layer of Xenopus laevis is formed upon egg activation by the binding of the cortical granule lectin (CGL) to its ligand in the egg extracellular matrix. Using Western blotting methods with biotinylated CGL as a probe, oviductal tissue extracts were examined to determine the site of origin of the CGL ligand. Three glycoprotein ligands of M_rs= > 250,000, 160,000, and 90,000 (reduced samples) were localized to the pars convoluta oviduct immediately posterior to the pars recta oviduct. The binding of CGL to these glycoproteins was inhibited in the presence of 200 mM galactose, but not with 200 mM mannose indicating a specific lectin interaction. The M_rs= > 250,000 and 90,000 glycoproteins were linked by disulfide bonds. In addition, these ligands were secreted from a more anterior region of the pars convoluta oviduct than the M_r=160,000 ligand. No binding of CGL was detected to pars recta secretory granule lysate components. The highest molecular weight CGL ligand seen in the pars convoluta corresponded to the CGL ligand in isolated fertilization envelopes. Thus, the CGL ligand involved in the formation of the fertilization layer is a product of the pars convoluta oviduct.     

The influence of residue 190 in the S1 site of trypsin-like serine proteases on substrate selectivity is universally conserved

We examined the influence of Ser/Ala190 in the S1 site on P1 substrate selectivity in several serine proteases. The impact of residue 190 on the selectivity was constant, regardless of differences in original selectivity or reactivity. Substrate binding in S1 was optimised in all wild-type enzymes, while the effects on k_cat depended on the combination of residue 190 and substrate. Mutagenesis of residue 190 did not affect the S2–S4 sites. Pronounced selectivity for arginine residues was coupled with low enzymatic activity, in particular in recombinant factor IXa. This is due to the dominance of the S1–P1 interaction over substrate binding in the S2–S4 sites.     

Crystallographic and mutational analyses of cystathionine β‐synthase in the H2S‐synthetic gene cluster in Lactobacillus plantarum

Cystathionine β‐synthase (CBS) catalyzes the formation of l ‐cystathionine from l ‐serine and l ‐homocysteine. The resulting l ‐cystathionine is decomposed into l ‐cysteine, ammonia, and α‐ketobutylic acid by cystathionine γ‐lyase (CGL). This reverse transsulfuration pathway, which is catalyzed by both enzymes, mainly occurs in eukaryotic cells. The eukaryotic CBS and CGL have recently been recognized as major physiological enzymes for the generation of hydrogen sulfide (H₂S). In some bacteria, including the plant‐derived lactic acid bacterium Lactobacillus plantarum, the CBS‐ and CGL‐encoding genes form a cluster in their genomes. Inactivation of these enzymes has been reported to suppress H₂S production in bacteria; interestingly, it has been shown that H₂S suppression increases their susceptibility to various antibiotics. In the present study, we characterized the enzymatic properties of the L. plantarum CBS, whose amino acid sequence displays a similarity with those of O‐acetyl‐l ‐serine sulfhydrylase (OASS) that catalyzes the generation of l ‐cysteine from O‐acetyl‐l ‐serine (l ‐OAS) and H₂S. The L. plantarum CBS shows l ‐OAS‐ and l ‐cysteine‐dependent CBS activities together with OASS activity. Especially, it catalyzes the formation of H₂S in the presence of l ‐cysteine and l ‐homocysteine, together with the formation of l ‐cystathionine. The high affinity toward l ‐cysteine as a first substrate and tendency to use l ‐homocysteine as a second substrate might be associated with its enzymatic ability to generate H₂S. Crystallographic and mutational analyses of CBS indicate that the Ala70 and Glu223 residues at the substrate binding pocket are important for the H₂S‐generating activity.     

ADAR2 A-->I editing: site selectivity and editing efficiency are separate events

ADAR enzymes, adenosine deaminases that act on RNA, form a family of RNA editing enzymes that convert adenosine to inosine within RNA that is completely or largely double-stranded. Site-selective A→I editing has been detected at specific sites within a few structured pre-mRNAs of metazoans. We have analyzed the editing selectivity of ADAR enzymes and have chosen to study the naturally edited R/G site in the pre-mRNA of the glutamate receptor subunit B (GluR-B). A comparison of editing by ADAR1 and ADAR2 revealed differences in the specificity of editing. Our results show that ADAR2 selectively edits the R/G site, while ADAR1 edits more promiscuously at several other adenosines in the double-stranded stem. To further understand the mechanism of selective ADAR2 editing we have investigated the importance of internal loops in the RNA substrate. We have found that the immediate structure surrounding the editing site is important. A purine opposite to the editing site has a negative effect on both selectivity and efficiency of editing. More distant internal loops in the substrate were found to have minor effects on site selectivity, while efficiency of editing was found to be influenced. Finally, changes in the RNA structure that affected editing did not alter the binding abilities of ADAR2. Overall these findings suggest that binding and catalysis are independent events.     

Molecular Basis for Galactosylation of Core Fucose Residues in Invertebrates: IDENTIFICATION OF CAENORHABDITIS ELEGANS N-GLYCAN CORE ��1,6-FUCOSIDE ��1,4-GALACTOSYLTRANSFERASE GALT-1 AS A MEMBER OF A NOVEL GLYCOSYLTRANSFERASE FAMILY*

Galectin CGL2 from the ink cap mushroom Coprinopsis cinerea displays toxicity toward the model nematode Caenorhabditis elegans. A mutation in a putative glycosyltransferase-encoding gene resulted in a CGL2-resistant C. elegans strain characterized by N-glycans lacking the β1,4-galactoside linked to the α1,6-linked core fucose. Expression of the corresponding GALT-1 protein in insect cells was used to demonstrate a manganese-dependent galactosyltransferase activity. In vitro, the GALT-1 enzyme showed strong selectivity for acceptors with α1,6-linked N-glycan core fucosides and required Golgi- dependent modifications on the oligosaccharide antennae for optimal synthesis of the Gal-β1,4-fucose structure. Phylogenetic analysis of the GALT-1 protein sequence identified a novel glycosyltransferase family (GT92) with members widespread among eukarya but absent in mammals.     

Structural and biochemical studies of the substrate selectivity of carnitine acetyltransferase

Carnitine acyltransferases catalyze the exchange of acyl groups between coenzyme A (CoA) and carnitine. They have important roles in many cellular processes, especially the oxidation of long-chain fatty acids, and are attractive targets for drug discovery against diabetes and obesity. These enzymes are classified based on their substrate selectivity for short-chain, medium-chain, or long-chain fatty acids. Structural information on carnitine acetyltransferase suggests that residues Met-564 and Phe-565 may be important determinants of substrate selectivity with the side chain of Met-564 located in the putative binding pocket for acyl groups. Both residues are replaced by glycine in carnitine palmitoyltransferases. To assess the functional relevance of this structural observation, we have replaced these two residues with small amino acids by mutagenesis, characterized the substrate preference of the mutants, and determined the crystal structures of two of these mutants. Kinetic studies confirm that the M564G or M564A mutation is sufficient to increase the activity of the enzyme toward medium-chain substrates with hexanoyl-CoA being the preferred substrate for the M564G mutant. The crystal structures of the M564G mutant, both alone and in complex with carnitine, reveal a deep binding pocket that can accommodate the larger acyl group. We have determined the crystal structure of the F565A mutant in a ternary complex with both the carnitine and CoA substrates at a 1.8-A resolution. The F565A mutation has minor effects on the structure or the substrate preference of the enzyme.     

Mutagenesis of the phosphate-binding pocket of KDPG aldolase enhances selectivity for hydrophobic substrates

Narrow substrate specificities often limit the use of enzymes in biocatalysis. To further the development of Escherichia coli 2-keto-3-deoxy-6-phosphogluconate (KDPG) aldolase as a biocatalyst, the molecular determinants of substrate specificity were probed by mutagenesis. Our data demonstrate that S184 is located in the substrate-binding pocket and interacts with the phosphate moiety of KDPG, providing biochemical support for the binding model proposed on the basis of crystallographic data. An analysis of the substrate selectivity of the mutant enzymes indicates that alterations to the phosphate-binding site of KDPG aldolase changes the substrate selectivity. We report mutations that enhance catalysis of aldol cleavage of substrates lacking a phosphate moiety and demonstrate that electrophile reactivity correlates with the hydrophobicity of the substituted side chain. These mutations improve the selectivity for unnatural substrates as compared to KDPG by up to 2000-fold. Furthermore, the S184L KDPG aldolase mutant improves the catalytic efficiency for the synthesis of a precursor for nikkomycin by 40-fold, making it a useful biocatalyst for the preparation of fine chemicals.     

Predicting substrate selectivity between UGT1A9 and UGT1A10 using molecular modelling and molecular dynamics approach

Uridine 5′-diphospho-glucuronosyltransferase-1A9 (UGT1A9) expressed in the liver, shows good sequence identity with UGT1A10, expressed in the intestine. Both uridine 5′-diphospho-glucuronosyltransferase (UGT) isoforms show comprehensive overlapping substrate selectivity but there are differences in stereoselectivity, regiospecificity and rate of glucuronidation of the substrates. Multiple sequence alignment analyses of UGT1A9 and UGT1A10 showed that 13% of the residues in N-terminal domain (NTD) are non-identical between them. Herein, authors attempted homology modelling of UGT1A9 and UGT1A10 and validation using software tools and reported mutagenic studies. A molecular docking study of the known substrates is performed on UGT1A9 and UGT1A10 homology models. The non-identical N-terminal residues ranging from 111 to 117 in UGT1A9 and UGT1A10 were identified to play a central role in the substrate selectivity. However, substrate binding is performed by Ser111, Gly115 and Leu117 in UGT1A10 and Gly111, Asp115 and Phe117 in UGT1A9. This study reports new residues in NTD, showing interaction with uridine 5′-diphospho-glucuronic acid which binds with C-terminal domain. Further, molecular dynamics simulations were carried out to study the role of non-identical residues in substrate identification. The study demonstrates the folding of the UGT enzyme, particularly, helix-loop-helix transition and movement of Nα3-2 helix, in response to substrate and co-substrate binding.     

Transition-state characterization: a new approach combining inhibitor analogues and variation in enzyme structure.

A new strategy of potentially broad application for probing transition-state (TS) analogy in enzymatic systems is described in this paper. The degree to which a series of phosphonate inhibitors act as TS analogues of rat carboxypeptidase A1 has been determined for the wild-type enzyme, for the R127K, R127M, and R127A mutants, and for the R127A mutant in the presence of 0.5 M guanidine hydrochloride. The impact that the mutations have on the inverse second-order rate constants (Km/kcat) for substrate hydrolysis is mirrored by the effect on the inhibition constants (Ki) for the corresponding phosphonate inhibitors. These results demonstrate that the phosphonate moiety mimics some of the electronic as well as the geometric characteristics of the TS. A similar but distinctly separate correlation is observed for tripeptide analogues in comparison to analogues of the dipeptide Cbz-Gly-Phe, reflecting an anomalous mode of binding for the latter system. The selective rate increases and corresponding enhancement in inhibitor binding observed on addition of 0.5 M guanidine hydrochloride to the R127A mutant indicate that the exogenous cation can assume the role played by Arg-127 in stabilizing the TS and in providing substrate selectivity at the P2 position.