Purification, characterization and reaction mechanism of novel arylsulfotransferase obtained from an anaerobic bacterium of human intestine

A novel type of arylsulfotransferase was purified from Eubacterium A-44, one of the predominant bacteria of human intestine. The enzyme (Mr 315 000) was composed of four identical subunits (Mr 80 000) whose N-terminal amino acids were arginine. pI and optimal pH of the enzyme were 3.9 and 8-9, respectively. The apparent Km for p-nitrophenylsulfate using tyramine as an acceptor substrate and that for tyramine using p-nitrophenylsulfate as a donor substrate were determined to be 0.104 mM and 3.5 mM, respectively. The reaction mechanism of the enzyme was proposed as follows: a donor substrate, p-nitrophenyl [35S]sulfate, combines a histidine residue of the enzyme active site with concomitant release of a phenolic compound, p-nitrophenol. The sulfate group of the histidine residue transfers to a tyrosine group, and then to an acceptor with the binding of another donor to the histidine residue.     

Molecular cloning of the arylsulfate sulfotransferase gene and characterization of its product from Enterobacter amnigenus AR-37

The gene encoding the Enterobacter amnigenus AR-37 arylsulfate sulfotransferase (ASST) was cloned, sequenced, and expressed in Escherichia coli NM522. Sequencing led to the identification of three contiguous open reading frames (ORFs) on the same strand. Based on amino acid sequence homology, ORF1, ORF2, and ORF3 are designated astA, dsbA, and dsbB, respectively. A multiple sequence alignment revealed conserved regions in ASST. An N-terminal amino acid sequence analysis of the purified ASST from E. coli NM522 (pEAST72) showed that it is subject to N-terminal processing. The specific activity of purified ASST is 436.5 U/mg of protein. The enzyme is a monomeric protein with a molecular mass of 64 kDa. Using phenol as an acceptor substrate, 4-methylumbelliferyl sulfate is the best donor substrate, followed by beta-naphthyl sulfate, p-nitrophenyl sulfate (PNS), and alpha-naphthyl sulfate. For PNS, alpha-naphthol is the best acceptor substrate, followed by phenol, resorcinol, p-acetaminophen, tyramine, and tyrosine. The enzyme has a different acceptor specificity than the enzyme purified from Eubacterium A-44. It is similar to Klebsiella K-36 and Haemophilus K-12. The apparent K(m) values for PNS using phenol as an acceptor and for phenol using PNS as a donor are 0.163 and 0.314 mM, respectively. The pI and optimum pH are 6.1 and 9.0, respectively.     

Purification and characterization of novel sulfotransferase obtained from Klebsiella K-36, an intestinal bacterium of rat.

A novel type of sulfotransferase was purified from Klebsiella K-36, an intestinal bacterium of rat. The enzyme (M(r) 160,000) is composed of two subunits (M(r) 73,000) with pI and optimal pH values of 5.3 and 10-10.5, respectively. The apparent Km for PNS (p-nitrophenyl sulfate) using phenol as an acceptor and that for phenol using PNS as a donor substrate were determined to be 0.11 and 0.66 mM, respectively. The enzyme is activated by magnesium ion and inhibited by EDTA.     

A novel type of arylsulfotransferase

Arylsulfotransferase catalyzes the transfer of a sulfate group from 3-phosphoadenosine-5-phosphosulfate (PAPS) to a phenolic acceptor substrate. We discovered a novel type of sulfotransferase from an anaerobic bacterium of human intestine, Eubacterium A-44. In the bacterial enzyme PAPS did not serve as a donor and all alcohols did not as acceptors. The new arylsulfotransferase was purified 185-fold from a crude extract of sonicated bacteria to homogeneity. The enzyme (MW 315 kd) was composed of four identical subunits (MW 80 kd) whose N-terminal amino acid was arginine, and its optimal pH and pI were 8–9 and 3.9, respectively. The enzyme catalyzed stoichiometric transfer of a sulfate group from a phenol sulfate ester to other phenols, with strict specificity. With tyramine as an acceptor, p-acetylphenyl sulfate was the best donor, followed by 4-methylumbelliferyl sulfate and p-nitrophenyl sulfate. With p-nitrophenyl sulfate as a donor, naphthol was the best acceptor, followed by estradiol, phenol, tyrosine methylester, tyramine, and epinephrine in decreasing order. Only the 4-position of catecholamines was specifically sulfated. Naturally occurring phenolic compounds, such as flavone, chalcone, and xanthone, were sulfated as well. Tyrosine-containing peptides were enzymatically sulfated: enkephalin, LH-RH, vasopressin, angiotensins, proctorin, CCK-8, and phyllocaerulein were sulfated with high yields. The novel sulfotransferase is expected to be applicable to enzymatic O-sulfation of tyrosine-containing hormones. The ³⁵S-labeled sulfate group from (³⁵S)p-nitrophenyl sulfate was incorporated into a tyrosyl residue at the active site of the enzyme (2 mole ³⁵S/mole of enzyme). The enzyme was inactivated by diethylpyrocarbamate and TLCK, chemical modifying agents for a histidyl residue. The reaction mechanism of arylsulfotransferase was proposed as follows: a donor substrate combines a histidyl residue with concomitant release of a phenolic compound. The sulfate group of the histidyl residue transfers to a tyrosyl residue, and then to an acceptor with the binding of another donor substrate to the histidyl residue.This article was presented during the proceedings of the International Conference on Macromolecular Structure and Function, held at the National Defence Medical College, Tokorozawa, Japan, December 1985.     

Kinetic mechanism and identification of the active site tyrosine residue in Enterobacter amnigenus arylsulfate sulfotransferase.

Bacterial arylsulfate sulfotransferase (ASST) catalyzes the transfer of a sulfate group from a phenyl sulfate ester to a phenolic acceptor. The kinetic mechanism of Enterobacter amnigenus ASST was determined. Plots of 1/v versus 1/[substrate (A)] at different fixed substrate (B) concentrations gave a series of parallel lines. One of the reaction products, p-nitrophenol, inhibited the enzyme noncompetitively with respect to p-nitrophenyl sulfate, but competitively to alpha-naphthol. These results correspond to a ping pong bi bi mechanism. By site-directed mutagenesis, we substituted each conserved tyrosine residue with phenylalanine. Among the mutants, Y123F showed severely reduced catalytic activity. We conclude that Tyr 123 is an essential active site residue. A mechanistic hypothesis is presented to account for these observations.     

Enzymatic attachment of glycosaminoglycan chain to peptide using the sugar chain transfer reaction with endo-beta-xylosidase.

Endo-beta-xylosidase from the mid-gut gland of the molluscus Patinopecten is an endo-type glycosidase that hydrolyzes the xylosyl serine linkage between a core protein and a glycosaminoglycan (GAG) chain, releasing the intact GAG chain from proteoglycan. In this study, we investigated GAG chain transfer activity of this enzyme, in order to develop a method for attaching GAG chains to peptide. Peptidochondroitin sulfate (molecular mass of sugar chain, 30 kDa) from bovine tracheal cartilage as a donor and butyloxycarbonyl-leucyl-seryl-threonyl-arginine-(4-methylcoumaryl-7-amide) as an acceptor were incubated with endo-beta-xylosidase. As a result, a reaction product with the same fluorescence as the acceptor peptide was observed. High pressure liquid chromatography analysis, cellulose acetate membrane electrophoresis, and enzymatic digestion showed that this reaction product had the chondroitin sulfate (ChS) from the donor. Furthermore, the acceptor peptide was released from this reaction product after hydrolysis by endo-beta-xylosidase. Therefore, it was confirmed that the ChS chain released from the donor was transferred to the acceptor peptide by the GAG chain transfer reaction of endo-beta-xylosidase. The optimal pH for hydrolysis by this enzyme was found to be about 4.0, whereas that for this reaction was about 3.0. Not only the ChS but also the dermatan sulfate and the heparan sulfate were transferred to the acceptor peptide by this reaction. By using this reaction, the GAG chain could be attached to the peptide in one step. The GAG chain transfer reaction of endo-beta-xylosidase should be a significant glycotechnological tool for the artificial synthesis of proteoglycan.     

NMR spectroscopic and molecular modeling investigations of the trans-sialidase from Trypanosoma cruzi

Nuclear magnetic resonance (NMR) spectroscopy was used to investigate the transfer of sialic acid from a range of sialic acid donor compounds to acceptor molecules, catalyzed by Trypanosoma cruzi trans-sialidase (TcTS). We demonstrate here that NMR spectroscopy is a powerful tool to monitor the trans-sialidase enzyme reaction for a variety of donor and acceptor molecules. The hydrolysis or transfer reactions that are catalyzed by TcTS were also investigated using a range of N-acetylneuraminosyl-based donor substrates and asialo acceptor molecules. These studies showed that the synthetic N-acetylneuraminosyl donor 4-methylumbelliferyl alpha-d-N-acetylneuraminide (MUN) is hydrolyzed by the enzyme approximately 3-5 times faster than either the disaccharide Neu5Acalpha(2,3)Galbeta1Me or the trisaccharide Neu5Acalpha(2,3)Lacbeta1Me. In the transfer reaction, we show that Neu5Acalpha(2,3)Lacbeta1Me is the most favorable substrate for TcTS and is a better substrate than the naturally-occurring N-acetylneuraminosyl donor alpha1-acid glycoprotein. In the case of MUN as the donor molecule, the transfer of Neu5Ac to different acceptors is significantly slower than when other N-acetylneuraminosyl donors are used. We hypothesize that when MUN is bound by the enzyme, the orientation and steric bulk of the umbelliferyl aglycon moiety may restrict the access for the correct positioning of an acceptor molecule. AutoDock studies support our hypothesis and show that the umbelliferyl aglycon moiety undergoes a strong pi-stacking interaction with Trp-312. The binding properties of TcTS towards acceptor (lactose) and donor substrate (Neu5Ac) molecules have also been investigated using saturation transfer difference (STD) NMR experiments. These experiments, taken together with other published data, have clearly demonstrated that lactose in the absence of other coligands does not bind to the TcTS active site or other binding domains. However, in the presence of the sialic acid donor, lactose (an asialo acceptor) was observed by NMR spectroscopy to interact with the enzyme's active site. The association of the asialo acceptor with the active site is an absolute requirement for the transfer reaction to proceed.     

A highly sensitive fluorometric assay for sialyltransferase activity using CMP-9-fluoresceinyl-NeuAc as donor

This paper presents a very sensitive fluorometric assay for sialyltransferase activity based on the transfer of 5-acetamido-9-deoxy-9-fluoresceinylthioure-idoneuraminic acid onto distinct glycoproteins, thus allowing determination of acceptor specificities. Acceptor protein-bound fluorescence was quantified after gel filtration which separated fluorescent sialoglycoprotein from the fluorescence-labeled CMP-glycoside donor. Kinetic constants obtained for five different purified sialyltransferases indicated that CMP-9-fluoresceinyl-NeuAc was a suitable donor substrate for each enzyme, affording low Km values and Vmax values comparable in magnitude (15-100%) to that obtained with the parent CMP-NeuAc. Sensitivity was enhanced 200- to 1000-fold compared to the radiometric sialyltransferase assay as it is used routinely. The method was applied to determination of the kinetic properties of purified rat liver alpha 2,6-sialyltransferase with four separate glycoprotein acceptors differing in glycan structure, employing very small amounts of donor, acceptor, and enzyme, and to the study of sialyltransferase activity of the human promyelocytic cell line HL-60 toward three different acceptors.     

Structural basis for acceptor substrate recognition of a human glucuronyltransferase, GlcAT-P, an enzyme critical in the biosynthesis of the carbohydrate epitope HNK-1

The HNK-1 carbohydrate epitope is found on many neural cell adhesion molecules. Its structure is characterized by a terminal sulfated glucuronyl acid. The glucuronyltransferases, GlcAT-P and GlcAT-S, are involved in the biosynthesis of the HNK-1 epitope, GlcAT-P as the major enzyme. We overexpressed and purified the recombinant human GlcAT-P from Escherichia coli. Analysis of its enzymatic activity showed that it catalyzed the transfer reaction for N-acetyllactosamine (Galbeta1-4GlcNAc) but not lacto-N-biose (Galbeta1-3GlcNAc) as an acceptor substrate. Subsequently, we determined the first x-ray crystal structures of human GlcAT-P, in the absence and presence of a donor substrate product UDP, catalytic Mn(2+), and an acceptor substrate analogue N-acetyllactosamine (Galbeta1-4GlcNAc) or an asparagine-linked biantennary nonasaccharide. The asymmetric unit contains two independent molecules. Each molecule is an alpha/beta protein with two regions that constitute the donor and acceptor substrate binding sites. The UDP moiety of donor nucleotide sugar is recognized by conserved amino acid residues including a DXD motif (Asp(195)-Asp(196)-Asp(197)). Other conserved amino acid residues interact with the terminal galactose moiety of the acceptor substrate. In addition, Val(320) and Asn(321), which are located on the C-terminal long loop from a neighboring molecule, and Phe(245) contribute to the interaction with GlcNAc moiety. These three residues play a key role in establishing the acceptor substrate specificity.     

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

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

Intermembrane phospholipid transfer mediated by cell-free extracts of Rhodopseudomonas sphaeroides.

The transfer of phospholipids between two membrane substrates catalyzed by a soluble protein fraction from Rhodopseudomonas sphaeroides has been demonstrated. The assay employs purified intracytoplasmic membrane (ICM) vesicles derived from cells of R. sphaeroides grown on [3H]acetate as the phospholipid donor substrate and phosphatidylcholine (70%)/phosphatidylethanolamine (30%) unilamellar liposomes containing [14C]triolein, a nontransferable marker, as the acceptor substrate for transferred phospholipids. Incubation of these two membrane substrates with a 40 to 80% (NH4)2SO4 protein fraction from R. sphaeroides results in the transfer of tritium-labeled ICM phospholipids to the acceptor membrane substrate. Upon completion of the incubation period, the donor ICM vesicles are quantitatively separated from the acceptor liposomes by precipitation with antibody prepared against whole, purified ICM vesicles. Phospholipid transfer is linear with respect to time and protein concentration, is inhibited by trypsin and heat, and shows an absolute dependence upon the presence of acceptor liposomes and the 40 to 80% (NH4)2SO4 protein fraction. Control experiments indicate that no fusion of the donor and acceptor membrane occurs during the incubation period and that, following prolonged incubation there is no detectable degradation of the labeled lipid components. Preliminary data on the phospholipid specificity of the transfer reaction is also presented.     

Use of N-acetyl-2'-O-methyllactosamine as a specific acceptor for the determination of alpha-L-(1----3)-fucosyltransferase in human serum

A synthetic substrate, N-acetyl-2'-O-methyllactosamine, was employed as a specific acceptor for alpha-L-(1----3)-fucosyltransferase from human serum. The fucosyl linkage of the product from this substrate was characterized by hydrolysis with a specific alpha-L-(1----3)/(1----4)-fucosidase. Using this acceptor, the pH optimum for the serum alpha-L-(1----3)-fucosyltransferase was 6.5. The enzyme was activated by Mn2+ or Mg2+ ions and was inhibited by EDTA. The apparent Km for this enzyme using N-acetyl-2'-O-methyllactosamine was 20.4 mM and Vmax was 5.6 pmol/h/ml serum.     

Crystal structure of Vibrionaceae Photobacterium sp. JT-ISH-224 alpha2,6-sialyltransferase in a ternary complex with donor product CMP and acceptor substrate lactose: catalytic mechanism and substrate recognition

Sialyltransferases are a family of glycosyltransferases that catalyze the transfer of N-acetylneuraminic acid residues from cytidine monophosphate N-acetylneuraminic acid (CMP-NeuAc) as a donor substrate to the carbohydrate groups of glycoproteins and glycolipids as acceptor substrates. We determined the crystal structure of Delta16psp26ST, the N-terminal truncated form of alpha2,6-sialyltransferase from Vibrionaceae Photobacterium sp. JT-ISH-224, complexed with a donor product CMP and an acceptor substrate lactose. Delta16psp26ST has three structural domains. Domain 1 belongs to the immunoglobulin-like beta-sandwich fold, and domains 2 and 3 form the glycosyltransferase-B structure. The CMP and lactose were bound in the deep cleft between domains 2 and 3. In the structure, only Asp232 was within hydrogen-binding distance of the acceptor O6 carbon of the galactose residue in lactose, and His405 was within hydrogen-binding distance of the phosphate oxygen of CMP. Mutation of these residues greatly decreased the activity of the enzyme. These structural and mutational results indicated that Asp232 might act as a catalytic base for deprotonation of the acceptor substrate, and His405 might act as a catalytic acid for protonation of the donor substrate. These findings are consistent with an in-line-displacement reaction mechanism in which Delta16psp26ST catalyzes the inverting transfer reaction. Unlike the case with multifunctional sialyltransferase (Delta24PmST1) complexed with CMP and lactose, the crystal structure of which was recently reported, the alpha2,6 reaction specificity of Delta16psp26ST is likely to be determined by His123.     

日本根霉IFO5318胞外β—葡萄糖苷酶的纯化及部分特性

采用硫酸铵沉淀及柱层析等步骤纯化了日本根霉IFO5318的β—葡萄糖苷酶,回收率为22％。该酶分子量约为4.0×10~5,由四个相同大小的亚基组成;最适反应温度55℃,最适反应pH5.5;对热较敏感,但能在较大的pH范围内保持稳定。用对硝基苯基—β-D-吡喃葡糖苷为底物,测得的K_m和V_(max)值分别为0.825mg·ml~(-1)和135.4μmol·min~(-1)·mg~(-1)。该酶对纤维二糖的水解能力最强,SDS、Fe~(3 )、Hg~2 )等对酶活力有抑制作用。     

Impact of donor binding on polymerization catalyzed by KfoC by regulating the affinity of enzyme for acceptor

BackgroundCurrently marketed chondroitin sulfate isolated from animal sources and structurally quite heterogeneous. Synthesis of structurally defined chondroitin sulfate is highly desired. The capsular polysaccharide from Escherichia coli strain K4 is similar to chondroitin, and its biosynthesis requires a chondroitin polymerase (KfoC). The essential step toward de novo enzymatic synthesis of chondroitin sulfate, synthesis of chondroitin, could be achieved by employing this enzyme.MethodsStructurally defined acceptors and donor-sugars were prepared by chemoenzymatic approaches. In addition, surface plasmon resonance was employed to determine the binding affinities of individual substrates and donor–acceptor pairs for KfoC.ResultsKfoC has broad donor substrate specificity and acceptor promiscuity, making it an attractive tool enzyme for use in structurally-defined chimeric glycosaminoglycan oligosaccharide synthesis in vitro. In addition, the binding of donor substrate molecules regulated the affinity of KfoC for acceptors, then influenced the glycosyl transferase reaction catalyzed by this chondroitin polymerase.Conclusion and general significanceThese results assist in the development of enzymatic synthesis approaches toward chimeric glycosaminoglycan oligosaccharides and designing future strategies for directed evolution of KfoC in order to create mutants toward user-defined goals.     

Purification and properties of gamma-glutamyltranspeptidase from Proteus mirabilis.

gamma-Glutamyltranspeptidase was purified ca. 15,200-fold from cell-free extracts of Proteus mirabilis to electrophoretic homogeneity and then crystallized. The enzyme has an estimated molecular weight of 80,000 and consists of two different subunits with molecular weights of ca. 47,000 and 28,000. The purified enzyme catalyzed hydrolysis and transpeptidation of various gamma-glutamyl compounds, including the oxidized and reduced forms of glutathione, gamma-glutamyl compounds of L-phenylalanine, L-tyrosine, L-histidine, L-alpha-aminobutyrate, L-leucine, and p-nitroaniline. Glycylglycine, L-phenylalanine, L-methionine, L-histidine, L-tryptophan, and L-isoleucine were good acceptors of the gamma-glutamyl moiety in the transpeptidation reaction. Km values for gamma-glutamyl compounds were on the order of 10(-4) to 10(-5) M, and those for acceptor peptides and amino acids were on the order of 10(-2) to 10(-3) M. The enzyme was inhibited by L-serine plus borate and 6-diazo-5-oxo-L-norleucine, which are inhibitors of gamma-glutamyltranspeptidases isolated from mammals. Various amino acids alone were found to inhibit the transpeptidation competitively with a gamma-glutamyl donor. Kinetic analysis suggested that the reaction sequence of substrate binding and product release proceeds according to a ping pong bi bi mechanism.     

Crystal structure of beta 1,3-glucuronyltransferase I in complex with active donor substrate UDP-GlcUA

Beta1,3-glucuronyltransferase (GlcAT-I) is an essential enzyme involved in heparan sulfate and chondroitin sulfate biosynthesis. GlcAT-I is an inverting glycosyltransferase that catalyzes the transfer of glucuronic acid (GlcUA) to the common growing linker region Galbeta1-3Galbeta1-4Xyl that is attached to a serine side chain of a core protein. Previously the structure of GlcAT-I has been solved in the presence of the donor product UDP and an acceptor analog Galbeta1-3Galbeta1-4Xyl (Pedersen, L. C., Tsuchida, K., Kitagawa, H., Sugahara, K., Darden, T. A. & Negishi, M. (2000) J. Biol. Chem. 275, 34580-34585). Here we report the x-ray crystal structure of GlcAT-I in complex with the complete donor UDP-GlcUA, thereby providing structures of an inverting glycosyltransferase in which both the complete donor and acceptor substrates are present in the active site. This structure supports the in-line displacement reaction mechanism previously proposed. It also provides information on the essential amino acid residues that determine donor substrate specificity.     

Continuous fluorometric assay of phenol sulfotransferase.

Phenol sulfotransferases (EC 2.8.2.1) catalyze the sulfation of the acceptor hydroxyl group using 3'-phosphoadenosine 5'-phosphosulfate (PAPS) as the donor substrate. Previous assays of these enzymes, which exhibit varied acceptor substrate specificities, have required termination of the catalysis followed by isolation and quantitation of formed sulfate ester. In this report, the sulfation of the fluorescent compound, resorufin, is investigated. Reaction of PAPS with resorufin, catalyzed by bovine lung phenol sulfotransferase, bleaches the emission of this acceptor at the pH of the reaction (pH 6.4 optimum). It is thereby possible to continuously record the sulfation reaction. Analysis of single progress curves by integrated replot can be used to determine the initial velocities and also indicates the formation of a product inhibitor, probably resorufin sulfate ester, with Ki less than Km. Sensitivity of the reaction is less than 1 pmol/min. The maximal rate of resorufin sulfation by the bovine lung enzyme is estimated at 57 nmol/mg/min, which is 10% of the rate with an optimal substrate 2-naphthol. This assay may be most sensitive for phenol sulfotransferases with optimal activities at greater than pH 6, due to the acid-base properties of resorufin (pK alpha 6), which becomes nonfluorescent upon protonation.     

Substrate recognition by β-ketoacyl-ACP synthases

β-Ketoacyl-ACP synthase (KAS) enzymes catalyze Claisen condensation reactions in the fatty acid biosynthesis pathway. These reactions follow a ping-pong mechanism in which a donor substrate acylates the active site cysteine residue after which the acyl group is condensed with the malonyl-ACP acceptor substrate to form a β-ketoacyl-ACP. In the priming KASIII enzymes the donor substrate is an acyl-CoA while in the elongating KASI and KASII enzymes the donor is an acyl-ACP. Although the KASIII enzyme in Escherichia coli (ecFabH) is essential, the corresponding enzyme in Mycobacterium tuberculosis (mtFabH) is not, suggesting that the KASI or II enzyme in M. tuberculosis (KasA or KasB, respectively) must be able to accept a CoA donor substrate. Since KasA is essential, the substrate specificity of this KASI enzyme has been explored using substrates based on phosphopantetheine, CoA, ACP, and AcpM peptide mimics. This analysis has been extended to the KASI and KASII enzymes from E. coli (ecFabB and ecFabF) where we show that a 14-residue malonyl-phosphopantetheine peptide can efficiently replace malonyl-ecACP as the acceptor substrate in the ecFabF reaction. While ecFabF is able to catalyze the condensation reaction when CoA is the carrier for both substrates, the KASI enzymes ecFabB and KasA have an absolute requirement for an ACP substrate as the acyl donor. Provided that this requirement is met, variation in the acceptor carrier substrate has little impact on the k(cat)/K(m) for the KASI reaction. For the KASI enzymes we propose that the binding of ecACP (AcpM) results in a conformational change that leads to an open form of the enzyme to which the malonyl acceptor substrate binds. Finally, the substrate inhibition observed when palmitoyl-CoA is the donor substrate for the KasA reaction has implications for the importance of mtFabH in the mycobacterial FASII pathway.     

Kinetic analysis using low-molecular mass xyloglucan oligosaccharides defines the catalytic mechanism of a Populus xyloglucan endotransglycosylase

Plant XETs [XG (xyloglucan) endotransglycosylases] catalyse the transglycosylation from a XG donor to a XG or low-molecular-mass XG fragment as the acceptor, and are thought to be important enzymes in the formation and remodelling of the cellulose-XG three-dimensional network in the primary plant cell wall. Current methods to assay XET activity use the XG polysaccharide as the donor substrate, and present limitations for kinetic and mechanistic studies of XET action due to the polymeric and polydisperse nature of the substrate. A novel activity assay based on HPCE (high performance capillary electrophoresis), in conjunction with a defined low-molecular-mass XGO {XG oligosaccharide; (XXXGXXXG, where G=Glcbeta1,4- and X=[Xylalpha1,6]Glcbeta1,4-)} as the glycosyl donor and a heptasaccharide derivatized with ANTS [8-aminonaphthalene-1,3,6-trisulphonic acid; (XXXG-ANTS)] as the acceptor substrate was developed and validated. The recombinant enzyme PttXET16A from Populus tremula x tremuloides (hybrid aspen) was characterized using the donor/acceptor pair indicated above, for which preparative scale syntheses have been optimized. The low-molecular-mass donor underwent a single transglycosylation reaction to the acceptor substrate under initial-rate conditions, with a pH optimum at 5.0 and maximal activity between 30 and 40 degrees C. Kinetic data are best explained by a ping-pong bi-bi mechanism with substrate inhibition by both donor and acceptor. This is the first assay for XETs using a donor substrate other than polymeric XG, enabling quantitative kinetic analysis of different XGO donors for specificity, and subsite mapping studies of XET enzymes.