Active site modulation in the N-acetylneuraminate lyase sub-family as revealed by the structure of the inhibitor-complexed Haemophilus influenzae enzyme

The N-acetylneuraminate lyase (NAL) sub-family of (beta/alpha)(8) enzymes share a common catalytic step but catalyse reactions in different biological pathways. Known examples include NAL, dihydrodipicolinate synthetase (DHDPS), d-5-keto-4-deoxyglucarate dehydratase, 2-keto-3-deoxygluconate aldolase, trans-o-hydroxybenzylidenepyruvate hydrolase-aldolase and trans-2'-carboxybenzalpyruvate hydratase-aldolase. Little is known about the way in which the three-dimensional structure of the respective active sites are modulated across the sub-family to achieve cognate substrate recognition. We present here the structure of Haemophilus influenzae NAL determined by X-ray crystallography to a maximum resolution of 1.60 A, in native form and in complex with three substrate analogues (sialic acid alditol, 4-deoxy-sialic acid and 4-oxo-sialic acid). These structures reveal for the first time the mode of binding of the complete substrate in the NAL active site. On the basis of the above structures, that of substrate-complexed DHDPS and sequence comparison across the sub-family we are able to propose a unified model for active site modulation. The model is one of economy, allowing wherever appropriate the retention or relocation of residues associated with binding common substrate substituent groups. Our structures also suggest a role for the strictly conserved tyrosine residue found in all active sites of the sub-family, namely that it mediates proton abstraction by the alpha-keto acid carboxylate in a substrate-assisted catalytic reaction pathway.     

Exploring the active site of benzaldehyde lyase by modeling and mutagenesis

Benzaldehyde lyase (BAL) is a thiamin diphosphate-dependent enzyme, which catalyzes the breakdown of (R)-benzoin to benzaldehyde. In essence, this is the reverse of the carboligation reaction catalyzed by benzoylformate decarboxylase (BFD). Here, we describe the first steps towards understanding the factors influencing BFD to form a CC bond under conditions wherein BAL will cleave the same bond. What are the similarities and differences between these two enzymes that result in the different catalytic activities? The X-ray structures of BFD and pyruvate decarboxylase (PDC) were used as templates for modeling benzaldehyde lyase. The model shows that a glutamine residue, Gln113, replaces the active site histidines of BFD and PDC. Replacement of the Gln113 by alanine or histidine reduced the value of k(cat) for lyase activity by more than 200-fold. The residues in BFD interacting with the phenyl ring of benzoylformate have similarly positioned counterparts in BAL but Ser26, the residue known to interact with the carboxylate group of benzoylformate, has been replaced by an alanine (Ala28). The BAL A28S variant exhibited 7% of WT activity in the BAL assay but, in the most intriguing result, this variant was able to catalyze the decarboxylation of benzoylformate. Conversely, the BFD S26A variant was unable to cleave benzoin.     

High-resolution crystal structure of Arthrobacter aurescens chondroitin AC lyase: an enzyme-substrate complex defines the catalytic mechanism

Chondroitin lyases (EC 4.2.2.4 and EC 4.2.2.5) are glycosaminoglycan-degrading enzymes that act as eliminases. Chondroitin lyase AC from Arthrobacter aurescens (ArthroAC) is known to act on chondroitin 4-sulfate and chondroitin 6-sulfate but not on dermatan sulfate. Like other chondroitin AC lyases, it is capable of cleaving hyaluronan. We have determined the three-dimensional crystal structure of ArthroAC in its native form as well as in complex with its substrates (chondroitin 4-sulfate tetrasaccharide, CS(tetra) and hyaluronan tetrasaccharide) at resolution varying from 1.25 A to 1.9A. The primary sequence of ArthroAC has not been previously determined but it was possible to determine the amino acid sequence of this enzyme from the high-resolution electron density maps and to confirm it by mass spectrometry. The enzyme-substrate complexes were obtained by soaking the substrate into the crystals for varying lengths of time (30 seconds to ten hours) and flash-cooling the crystals. The electron density map for crystals soaked in the substrate for as short as 30 seconds showed the substrate clearly and indicated that the ring of central glucuronic acid assumes a distorted boat conformation. This structure strongly supports the lytic mechanism where Tyr242 acts as a general base that abstracts the proton from the C5 position of glucuronic acid while Asn183 and His233 neutralize the charge on the glucuronate acidic group. Comparison of this structure with that of chondroitinase AC from Flavobacterium heparinum (FlavoAC) provides an explanation for the exolytic and endolytic mode of action of ArthroAC and FlavoAC, respectively.     

Chondroitin O-methyl ester: an unusual substrate for chondroitin AC lyase

Chondroitin O-methyl ester was depolymerized by chondroitin AC lyase (EC 4.2.2.5) from Flavobacterium heparinum. The major product isolated from the depolymerization reaction was found to be methyl alpha-L-threo-hex-4-enopyranosyluronate-(1-->4)-2-acetamido-2-deoxy-alpha,beta-D-galactopyranoside.     

Crystal structure of chondroitin AC lyase, a representative of a family of glycosaminoglycan degrading enzymes.

Glycosaminoglycans (GAGs), highly sulfated polymers built of hexosamine-uronic acid disaccharide units, are major components of the extracellular matrix, mostly in the form of proteoglycans. They interact with a large array of proteins, in particular of the blood coagulation cascade. Degradation of GAGs in mammalian systems occurs by the action of GAG hydrolases. Bacteria express a large number of GAG-degrading lyases that break the hexosamine-uronic acid bond to create an unsaturated sugar ring. Flavobacterium heparinum produces at least five GAG lyases of different specificity. Chondroitin AC lyase (chondroitinase AC, 75 kDa) is highly active toward chondroitin 4-sulfate and chondroitin-6 sulfate. Its crystal structure has been determined to 1.9 A resolution. The enzyme is composed of two domains. The N-terminal domain of approximately 300 residues contains mostly alpha-helices which form a doubly-layered horseshoe (a subset of the (alpha/alpha)6 toroidal topology). The approximately 370 residues long C-terminal domain is made of beta-strands arranged in a four layered beta-sheet sandwich, with the first two sheets having nine strands each. This fold is novel and has no counterpart in full among known structures. The sequence of chondroitinase AC shows low level of homology to several hyaluronate lyases, which likely share its fold. The shape of the molecule, distribution of electrostatic potential, the pattern of conservation of the amino acids and the results of mutagenesis of hyaluronate lyases, indicate that the enzymatic activity resides primarily within the N-terminal domain. The most likely candidate for the catalytic base is His225. Other residues involved in catalysis and/or substrate binding are Arg288, Arg292, Lys298 and Lys299.     

Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.     

Role of arginine 292 in the catalytic activity of chondroitin AC lyase from Flavobacterium heparinum

Chondroitin AC lyase (chondroitinase EC 4.2.2.5), an eliminase from Flavobacterium heparinum, cleaves chondroitin sulfate glycosaminoglycans (GAGs) at 1,4 glycosidic linkages between N-acetylgalactosamine and glucuronic acid residues. Cleavage occurs through beta-elimination in a random endolytic action pattern. Crystal structures of chondroitin AC lyase (wild type) complexed with oligosaccharides reveal a binding site within a narrow and shallow protein channel, suggesting several amino acids as candidates for the active site residues. Site-specific mutagenesis studies on residues within the active-site tunnel revealed that only the Arg to Ala 292 mutation (R292A) retained activity. Furthermore, structural data suggested that R292 was primarily involved in recognition of N-acetyl or O-sulfo moieties of galactosamine residues and did not directly participate in catalysis. The current study demonstrates that the R292A mutation affords approximately 10-fold higher K(m) values but no significant change in V(max), consistent with hypothesis that R292 is involved in binding the O-sulfo moiety of the saccharide residues. Change in chondroitin sulfate viscosity, as a function of its enzymatic cleavage, affords a shallower concave curve for the R292A mutant, suggesting its action pattern is neither purely random endolytic nor purely random exolytic. Product studies using gel electrophoresis confirm the altered action pattern of this mutant. Thus, these data suggest that the R292A mutation effectively reduces binding affinity, making it possible for the oligosaccharide chain, still bound after initial endolytic cleavage, to slide through the tunnel to the catalytic site for subsequent, processive, step-wise, exolytic cleavage.     

Scaffolding functions of arrestin-2 revealed by crystal structure and mutagenesis

Arrestin binding to activated, phosphorylated G protein-coupled receptors (GPCRs) represents a critical step in regulation of light- and hormone-dependent signaling. Nonvisual arrestins, such as arrestin-2, interact with multiple proteins for the purpose of propagating and terminating signaling events. Using a combination of X-ray crystallography, molecular modeling, mutagenesis, and binding analysis, we reveal structural features of arrestin-2 that may enable simultaneous binding to phosphorylated receptor, SH3 domains, phosphoinositides, and beta-adaptin. The structure of full-length arrestin-2 thus provides a uniquely oriented scaffold for assembly of multiple, diverse molecules involved in GPCR signal transduction.     

Insights into enzyme evolution revealed by the structure of methylaspartate ammonia lyase.

Methylaspartate ammonia lyase (MAL) catalyzes the magnesium-dependent reversible alpha,beta-elimination of ammonia from L-threo-(2S,3S)-3-methylaspartic acid to mesaconic acid. The 1.3 A MAD crystal structure of the dimeric Citrobacter amalonaticus MAL shows that each subunit comprises two domains, one of which adopts the classical TIM barrel fold, with the active site at the C-terminal end of the barrel. Despite very low sequence similarity, the structure of MAL is closely related to those of representative members of the enolase superfamily, indicating that the mechanism of MAL involves the initial abstraction of a proton alpha to the 3-carboxyl of (2S,3S)-3-methylasparic acid to yield an enolic intermediate. This analysis resolves the conflict that had linked MAL to the histidine and phenylalanine ammonia lyase family of enzymes.     

Composite active site of chondroitin lyase ABC accepting both epimers of uronic acid

Enzymes have evolved as catalysts with high degrees of stereospecificity. When both enantiomers are biologically important, enzymes with two different folds usually catalyze reactions with the individual enantiomers. In rare cases a single enzyme can process both enantiomers efficiently, but no molecular basis for such catalysis has been established. The family of bacterial chondroitin lyases ABC comprises such enzymes. They can degrade both chondroitin sulfate (CS) and dermatan sulfate (DS) glycosaminoglycans at the nonreducing end of either glucuronic acid (CS) or its epimer iduronic acid (DS) by a beta-elimination mechanism, which commences with the removal of the C-5 proton from the uronic acid. Two other structural folds evolved to perform these reactions in an epimer-specific fashion: (alpha/alpha)(5) for CS (chondroitin lyases AC) and beta-helix for DS (chondroitin lyases B); their catalytic mechanisms have been established at the molecular level. The structure of chondroitinase ABC from Proteus vulgaris showed surprising similarity to chondroitinase AC, including the presence of a Tyr-His-Glu-Arg catalytic tetrad, which provided a possible mechanism for CS degradation but not for DS degradation. We determined the structure of a distantly related Bacteroides thetaiotaomicron chondroitinase ABC to identify additional structurally conserved residues potentially involved in catalysis. We found a conserved cluster located approximately 12 A from the catalytic tetrad. We demonstrate that a histidine in this cluster is essential for catalysis of DS but not CS. The enzyme utilizes a single substrate-binding site while having two partially overlapping active sites catalyzing the respective reactions. The spatial separation of the two sets of residues suggests a substrate-induced conformational change that brings all catalytically essential residues close together.     

A novel elicitin necrotic site revealed by alpha-cinnamomin sequence and site-directed mutagenesis.

Elicitins are 10 kDa proteins secreted by Phytophthora fungi, that elicit resistance against certain plant pathogens. Various natural molecules, mutated recombinant elicitins and synthetic peptides were previously shown to differentially induce in tobacco leaf necrosis and defence genes, activities borne by several sites which were identified. We report a novel necrosis-determining residue at position 25, revealed by the comparison of the necrotic activity and sequence of alpha-cinnamomin with those of other known elicitins. Using a modified recombinant beta-cryptogein, expressed in Pichia pastoris, we show that the substitution of asparagine 25 by a serine leads to a significant enhancement of the necrotic activity.     

Development of an assay and determination of kinetic parameters for chondroitin AC lyase using defined synthetic substrates

Many techniques have been developed for the assay of polysaccharide lyases; however, none have allowed the measurement of defined and reproducible k(cat) and K(m) values due to the inhomogeneous nature of the polymeric substrates. We have designed three different substrates for chondroitin AC lyase from Flavobacterium heparinum that can be monitored by three different techniques: UV/Vis spectroscopy, fluorescence spectroscopy, and use of a fluoride ion-selective electrode. Each is a continuous assay, free from interferences caused by other components present in crude enzyme preparations, and allows meaningful and reproducible kinetic parameters to be determined. The development of these defined synthetic substrates has opened up a wide variety of mechanistic studies that can be performed to elucidate the detailed catalytic mechanism of this, and other, polysaccharide lyases. The application of these techniques, which include kinetic isotope effects and linear free energy analyses, was not possible with the previous polymeric substrates and will allow this relatively poorly understood class of polysaccharide-degrading enzymes to be studied mechanistically.     

Cartilage degradation by hyaluronate lyase and chondroitin ABC lyase: a MALDI-TOF mass spectrometric study

Matrix-assisted laser desorption ionization and time-of-flight mass spectrometry (MALDI-TOF MS) has been used to investigate degradation products of two selected polysaccharides of cartilage (chondroitin sulfate and hyaluronic acid). Testicular hyaluronate lyase and chondroitin ABC lyase were used for enzymic digestion of both polysaccharides as well as of cartilage specimens. Polysaccharide solutions and cartilage supernatants were assayed by positive and negative MALDI-TOF MS. Especially chondroitin ABC lyase produced high amounts of digestion products (unsaturated di- and tetrasaccharides) from polysaccharides as well as from cartilage, clearly monitored by MALDI-TOF MS. It is concluded that MALDI-TOF MS provides a precise and fast tool for the determination of oligosaccharides since no previous derivatization is required.     

Discontinuities in the topography of alcohol dehydrogenase-sodium dodecyl sulphate complexes revealed by mutagenesis and proteolysis.

Experiments utilizing proteolytic mapping of maize Alcohol dehydrogenase-1 protein (EC 1.1.1.1; ADH) showed that, in the presence of sodium dodecyl sulphate (SDS), two discrete areas of the protein molecule were hypersensitive to digestion with Staphylococcus aureus V8 proteinase. These areas were mapped to points 11 and 27 kDa along the 41 kDa polypeptide. Furthermore, ADH1 proteins isolated from the ethyl methanesulphonate-induced mutants U725 and W586 showed both a change in electrophoretic mobility in SDS gels, and an altered V8 proteinase digestion pattern. Protein from U725 migrated in SDS gels as though it was 2 kDa smaller than wild-type ADH protein and lacked the 11 kDa cleavage site. Protein from W586 lacked the 30 kDa cleavage site and migrated as if it was 3 kDa smaller than wild type. The possible relationships between proteinase cleavage sites, mutations and SDS gel mobilities are discussed.     

Virulent and nonvirulent Flavobacterium columnare colony morphologies: characterization of chondroitin AC lyase activity and adhesion to polystyrene

Aims: Colony morphology variants of fish pathogenic Flavobacterium columnare were studied to clarify the role of colony morphology change in the virulence of the bacterium. Typical rhizoid colony (Rz) variants are virulent and moderately adherent, nonrhizoid rough (R) colony variants are nonvirulent and highly adherent, and soft colony (S) variants are nonvirulent and poorly adherent. Methods and Results: Chondroitin AC lyase activity, adhesion to polystyrene at different temperatures and after modification of bacterial surface, and lipopolysaccharide (LPS) profiles of the variants were studied. The chondroitinase activity was significantly higher in the virulent, rhizoid variants than in the rough variants of the same strain. Temperature significantly increased the adhesion of rhizoid variants up to 20°C. Modification of bacterial surface suggested that adhesion molecules contain both carbohydrates and proteins. LPS did not differ between the variants of the same strain. Conclusions: The results suggest that in Fl. columnare both rhizoid colony morphology and high chondroitinase activity are needed for virulence and that temperature may promote the adhesion of the virulent variants to surfaces at fish farms. Significance and Impact of the Study: New information is produced on the virulence mechanisms of Fl. columnare and the reasons behind the survival of the bacterium at fish farms.     

Active site plasticity revealed from the structure of the enterobacterial N-ribohydrolase RihA bound to a competitive inhibitor

Background  

Pyrimidine-preferring N-ribohydrolases (CU-NHs) are a class of Ca²⁺-dependent enzymes that catalyze the hydrolytic cleavage of the N-glycosidic bond in pyrimidine nucleosides. With the exception of few selected organisms, their physiological relevance in prokaryotes and eukaryotes is yet under investigation.     

Active site proton delivery and the lyase activity of human CYP17A1

Cytochrome P450 CYP17A1 catalyzes a series of reactions that lie at the intersection of corticoid and androgen biosynthesis and thus occupies an essential role in steroid hormone metabolism. This multifunctional enzyme catalyzes the 17α-hydroxylation of Δ4- and Δ5-steroids progesterone and pregnenolone to form the corresponding 17α-hydroxy products through its hydroxylase activity, and a subsequent 17,20-carbon–carbon scission of pregnene-side chain produce the androgens androstenedione (AD) and dehydroepiandrosterone (DHEA). While the former hydroxylation reaction is believed to proceed through a conventional “Compound I” rebound mechanism, it has been suggested that the latter carbon cleavage is initiated by an iron-peroxy intermediate. We report on the role of Thr306 in CYP17 catalysis. Thr306 is a member of the conserved acid/alcohol pair thought to be essential for the efficient delivery of protons required for hydroperoxoanion heterolysis and formation of Compound I in the cytochromes P450. Wild type and T306A CYP17A1 self-assembled in Nanodiscs were used to quantitate turnover and coupling efficiencies of CYP17’s physiological Δ4- and Δ5-substrates. We observed that T306A co-incorporated in Nanodiscs with its redox partner cytochrome P450 oxidoreductase, coupled NADPH only by 0.9% and 0.7% compared to the wild type (97% and 22%) during the conversion of pregnenolone and progesterone, respectively, to the corresponding 17-OH products. Despite increased oxidation of pyridine nucleotide, hydroxylase activity was drastically diminished in the T306A mutant, suggesting a high degree of uncoupling in which reducing equivalents and protons are funneled into non-productive pathways. This is similar to previous work with other P450 catalyzed hydroxylation. However, catalysis of carbon–carbon bond scission by the T306A mutant was largely unimpeded by disruption of the CYP17A1 acid-alcohol pair. The unique response of CYP17A1 lyase activity to mutation of Thr306 is consistent with a reactive intermediate formed independently of proton delivery in the active site, and supports involvement of a nucleophilic peroxo-anion rather than the traditional Compound I in catalysis.