Substrate Specificities of Variants of Barley (1,3)- and (1,3;1,4)-ß-d-Glucanases Resulting from Mutagenesis and Segment Hybridization.

Barley (1,3;1,4)-ß-d-glucanase is believed to have evolved from an ancestral monocotyledon (1,3)-ß-d-glucanase, enabling the hydrolysis of (1,3;1,4)-ß-d-glucans in the cell walls of leaves and germinating grains. In the present study, we investigated the substrate specificities of variants of the barley enzymes (1,3;1,4)-ß-d-glucan endohydrolase [(1,3;1,4)-ß-d-glucanase] isoenzyme EII (HvEII) and (1,3)-ß-d-glucan endohydrolase [(1,3)-ß-d-glucanase] isoenzyme GII (HvGII) obtained by protein segment hybridization and site-directed mutagenesis. Using protein segment hybridization, we obtained three variants of HvEII in which the substrate specificity was that of a (1,3)-ß-d-glucanase and one variant that hydrolyzed both (1,3)-ß-d-glucans and (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3;1,4)-ß-d-glucans. Using substitutions of specific amino acid residues, we obtained one variant of HvEII that hydrolyzed both substrates. However, neither protein segment hybridization nor substitutions of specific amino acid residues gave variants of HvGII that could hydrolyze (1,3;1,4)-ß-d-glucans; the wild-type enzyme hydrolyzed only (1,3)-ß-d-glucans. Other HvEII and HvGII variants showed changes in specific activity and their ability to degrade the (1,3;1,4)-ß-d-glucans or (1,3)-ß-d-glucans to larger oligosaccharides. We also used molecular dynamics simulations to identify amino-acid residues or structural regions of wild-type HvEII and HvGII that interact with (1,3;1,4)-ß-d-glucans and (1,3)-ß-d-glucans, respectively, and may be responsible for the substrate specificities of the two enzymes.

Protein Kinase CK2α', More than a Backup of CK2α.

The serine/threonine protein kinase CK2 is implicated in the regulation of fundamental processes in eukaryotic cells. CK2 consists of two catalytic α or α' isoforms and two regulatory CK2ß subunits. These three proteins exist in a free form, bound to other cellular proteins, as tetrameric holoenzymes composed of CK2α2/ß2, CK2αα'/ß2, or CK2α'2/ß2 as well as in higher molecular forms of the tetramers. The catalytic domains of CK2α and CK2α' share a 90% identity. As CK2α contains a unique C-terminal sequence. Both proteins function as protein kinases. These properties raised the question of whether both isoforms are just backups of each other or whether they are regulated differently and may then function in an isoform-specific manner. The present review provides observations that the regulation of both CK2α isoforms is partly different concerning the subcellular localization, post-translational modifications, and aggregation. Up to now, there are only a few isoform-specific cellular binding partners. The expression of both CK2α isoforms seems to vary in different cell lines, in tissues, in the cell cycle, and with differentiation. There are different reports about the expression and the functions of the CK2α isoforms in tumor cells and tissues. In many cases, a cell-type-specific expression and function is known, which raises the question about cell-specific regulators of both isoforms. Another future challenge is the identification or design of CK2α'-specific inhibitors.

Polypeptide N-acetylgalactosaminyltransferase (GalNAc-T) isozyme surface charge governs charge substrate preferences to modulate mucin type O-glycosylation.

A large family of polypeptide N-acetylgalactosaminyltransferases (GalNAc-Ts) initiate mucin type O-glycosylation transferring α-GalNAc from a UDP-GalNAc donor to the hydroxyl groups of Ser and Thr residues of peptides and proteins, thereby defining sites of O-glycosylation. Mutations and differential expression of several GalNAc-Ts are associated with many disease states including cancers. The mechanisms by which these isozymes choose their targets and their roles in disease are not fully understood. We previously showed that the GalNAc-Ts possess common and unique specificities for acceptor type, peptide sequence and prior neighboring, and/or remote substrate GalNAc glycosylation. In the present study, the role of flanking charged residues was investigated using a library of charged peptide substrates containing the central -YAVTPGP- acceptor sequence. Eleven human and one bird GalNAc-T were initially characterized revealing a range of preferences for net positive, net negative, or unique combinations of flanking N- and/or C-terminal charge, correlating to each isozyme's different electrostatic surface potential. It was further found that isoforms with high sequence identity (>70%) within a subfamily can possess vastly different charge specificities. Enzyme kinetics, activities obtained at elevated ionic strength, and molecular dynamics simulations confirm that the GalNAc-Ts differently recognize substrate charge outside the common +/-3 residue binding site. These electrostatic interactions impact how charged peptide substrates bind/orient on the transferase surface, thus modulating their activities. In summary, we show the GalNAc-Ts utilize more extended surfaces than initially thought for binding substrates based on electrostatic, and likely other hydrophobic/hydrophilic interactions, furthering our understanding of how these transferases select their target.

Investigation of the effects of some pesticides on carbonic anhydrase isoenzymes.

The aim of this study was to investigate the inhibitory effects of some pesticides known to have harmful effects on human health on carbonic anhydrase isoenzymes. Therefore, carbonic anhydrase isoenzymes (hCA I and II) were purified from human erythrocytes. The isoenzymes were purified from human erythrocytes by using an affinity column that has the chemical structure of Sepharose-4B-4-(6-amino-hexyloxy)-benzenesulfonamide. The purity of the isoenzymes was checked by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDSPAGE). It was determined that the pesticides used in this study inhibit hCA I and hCA II isoenzymes at different levels in vitro. It was determined that the strongest inhibitor for the hCA I enzyme was Carbofuran (IC50 :6.52 µM; Ki : 3.58 µM) and the weakest one was 1-Naphtol (IC50 :16.55 µM; Ki : 14.4 µM) among these pesticides. It was also found that the strongest inhibitor for the hCA II enzyme was coumatetralil (IC50 :5.06 µM; Ki : 1.62 µM) and the weakest one was Dimethachlor (IC50 14.6 µM; Ki : 8.44 µM).

Synthesis of Stereoisomeric Simplified Analogs of Alotaketals toward the Elucidation of the Structural Requirements of Protein Kinase C Isozyme-Selective Binding.

A set of four stereoisomeric compounds were designed and synthesized as ligands of protein kinase C (PKC). The compounds were simplified analogs of the alotaketals, a class of natural products that were predicted to be ligands of PKC by computational screening. Bioassays revealed that the orientation of the alkyl side chain of the analogs was important for PKC binding and that the stereochemistry of the fused ring moiety influenced the PKC isozyme selectivity.

Biological activity and molecular docking studies of some N-phenylsulfonamides against cholinesterases and carbonic anhydrase isoenzymes.

In this research, a series of N-phenylsulfonamide derivatives (1-12) were designed, synthesized, and investigated for their inhibitory potencies against carbonic anhydrase isoenzymes I, II, and IX (hCA I, hCA II, and hCA IX) and cholinesterases (ChE), namely, acetylcholinesterase and butyrylcholinesterase. These compounds, whose inhibition potentials were evaluated for the first time, were characterized by spectroscopic techniques (1 H- and 13 C-NMR and FT-IR). CA isoenzyme inhibitors are significant therapeutic targets, especially owing to their preventive/activation potential in the therapy processes of some diseases such as cancer, osteoporosis, and glaucoma. On the other hand, Cholinesterase inhibitors are valuable molecules with biological importance that can be employed in the therapy process of Alzheimer's patients. The results showed that the tested molecules had enzyme inhibition activities ranging from 9.7 to 93.7 nM against these five metabolic enzymes. Among the tested molecules, the methoxy and the hydroxyl group-containing compounds 10, 11, and 12 exhibited more enzyme inhibition activities when compared to standard compounds acetazolamide, sulfapyridine, and sulfadiazine for CA isoenzymes and neostigmine for ChE, respectively. Of these three molecules, compound 12, which had a hydroxyl group in the para position in the aromatic ring, was determined to be the most active molecule against all enzymes. In silico work, molecular docking has also shown similar results and is consistent with the experimental data in the study. As a result, we can say that some of the tested molecules might be used as promising inhibitor candidates for further studies on this topic.

Purification and Biochemical Characterization of Two Anionic Peroxidase Isoenzymes from Raphanus sativus L. var niger Roots.

Peroxidase is a commonly used enzyme with a wide range of applications. Horseradish (Armoracia rusticana) is the most well-known source of peroxidase enzyme. Peroxidases extracted from other plant sources have also been proved as useful, sometimes even superior, comparing to traditional horseradish peroxidase (HRP). In the present study, two novel peroxidase isoenzymes were purified and characterized from Raphanus sativus L. var niger roots. Two anionic peroxidase isoenzymes were purified using diafiltration, ammonium sulfate precipitation, DEAE anion-exchange chromatography, and concanavalin A affinity chromatography. The heaviest anionic isoenzyme (isoenzyme A) has a MW of about 110 KD, and the other anionic isoenzyme (isoenzyme B) has a MW of 97 KD. Both isoenzymes have an optimum temperature of 40 °C, but the activity of isoenzyme B is much more dependent on temperature with a Q10 of 3.5, while isoenzyme A has a Q10 of 1.7. These isoenzymes showed great thermal stability at 37 °C and 4 °C. Isoenzyme A showed the highest activity at pH 5 and it was found to be more stable at pH 6, whereas isoenzyme B showed the highest activity at pH 6 and is more stable at pH 7. Isoenzyme A has a Km value of 10.63 mM and 0.043 mM, and isoenzyme B has a Km of 15.38 mM and 0.067 mM for 4-aminoantipyrine and H2O2, respectively. The isoenzymes purified from Raphanus sativus L. var niger offer excellent chemical and thermal stability, which encourages further studies on their suitability for biotechnological applications.

Starch synthases SSIIa and GBSSI control starch structure but do not determine starch granule morphology in the absence of SSIIIa and SSIVb.

KEY MESSAGE: High levels of two major starch synthases, SSIIa and GBSSI, in ss3a ss4b double mutant rice alter the starch structure but fail to recover the polygonal starch granule morphology. The endosperm starch granule is polygonal in wild-type rice but spherical in double mutant japonica rice lacking genes encoding two of the five major Starch synthase (SS) isozymes expressed in endosperm, SSIIIa and SSIVb. Japonica rice naturally has low levels of SSIIa and Granule-bound SSI (GBSSI). Therefore, introduction of active SSIIa allele and/or high-expressing GBSSI allele from indica rice into the japonica rice mutant lacking SS isozymes can help elucidate the compensatory roles of SS isozymes in starch biosynthesis. In this study, we crossed the ss3a ss4a double mutant japonica rice with the indica rice to generate three new rice lines with high and/or low SSIIa and GBSSI levels, and examined their starch structure, physicochemical properties, and levels of other starch biosynthetic enzymes. Lines with high SSIIa levels showed more SSI and SSIIa bound to starch granule, reduced levels of short amylopectin chains (7 ≤ DP ≤ 12), increased levels of amylopectin chains with DP > 13, and consequently higher gelatinization temperature. Lines with high GBSSI levels showed an increase in amylose content. The ADP-glucose content of the crude extract was high in lines with low or high SSIIa and low GBSSI levels, but was low in lines with high GBSSI. Addition of high SSIIa and GBSSI altered the starch structure and physicochemical properties but did not affect the starch granule morphology, confirming that SSIIIa and SSIVb are key enzymes affecting starch granule morphology in rice. The relationship among SS isozymes and its effect on the amount of substrate (ADP-glucose) is discussed.

Relative importance of branching enzyme isoforms in determining starch fine structure and physicochemical properties of indica rice.

KEY MESSAGE: Down-regulation of starch branching enzymes alters fine structure and starch properties, especially the B-type crystalline pattern and extremely high amylose content identified in the BEIIb-deficiency mutant in the indica rice. The relative importance of the starch branching enzymes in determining the molecular fine structure and starch functional properties were uncovered in this study. An indica rice, Guangluai 4 with high amylose content (AC) and high gelatinization temperature (GT) was used to generate the clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein-9 (Cas9) knockout lines. Five mutant lines were identified including be1-1, be1-2, be2a-1, be2a-2 and be2b-1, and analysis of western blot showed the CRISPR/Cas9 system was successful in inducing mutations in the targeted genes. AC of be2b-1 (34.1%) was greater than that of wild type (WT) (27.4%) and other mutants. Mutations of either BEI or BEIIa did not alter the starch crystallite pattern (A-type). The BEIIb deficiency caused an opaque endosperm phenotype, changed the crystallite pattern from A- to B-type, and dramatically increased the degree of ordered structure, the relative proportion of amylose chains and intermediate to long amylopectin chains, average chain length of amylopectin molecules as well as GT. The BEIIa deficiency had no effect on the proportion of amylose chains, the length of amylopectin intermediate-long chains, conclusion temperature and enthalpy of gelatinization. Down-regulation of BEI increased the proportion of shortest amylopectin chains (fa) but decreased the proportion of long amylopectin chains (fb2 and fb3), leading to a lower GT. It is concluded that the relative importance in determining starch fine structures and functionality was in the order of BEIIb > BEI > BEIIa. Our results provide new information for utilizations of BE-deficient mutants in rice quality breeding.

Oligomerization of Ca2+/calmodulin-dependent protein kinase kinase.

Ca2+/calmodulin-dependent protein kinase kinases (CaMKKα and ß) are regulatory kinases for multiple downstream kinases, including CaMKI, CaMKIV, PKB/Akt, and AMP-activated protein kinase (AMPK) through phosphorylation of each activation-loop Thr residue. In this report, we biochemically characterize the oligomeric structure of CaMKK isoforms through a heterologous expression system using COS-7 cells. Oligomerization of CaMKK isoforms was readily observed by treating CaMKK transfected cells with cell membrane permeable crosslinkers. In addition, His-tagged CaMKKα (His-CaMKKα) pulled down with FLAG-tagged CaMKKα (FLAG-CaMKKα) in transfected cells. The oligomerization of CaMKKα was confirmed by the fact that GST-CaMKKα/His-CaMKKα complex from transiently expressed COS-7 cells extracts was purified to near homogeneity by the sequential chromatography using glutathione-sepharose/Ni-sepharose and was observed in a Ca2+/CaM-independent manner by reciprocal pulldown assay, suggesting the direct interaction between monomeric CaMKKα. Furthermore, the His-CaMKKα kinase-dead mutant (D293A) complexed with FLAG-CaMKKα exhibited significant CaMKK activity, indicating the active CaMKKα multimeric complex. Collectively, these results suggest that CaMKKα can self-associate in the cells, constituting a catalytically active oligomer that might be important for the efficient activation of CaMKK-mediated intracellular signaling.

Glucomannan and beta-glucan degradation by Mytilus edulis Cel45A: Crystal structure and activity comparison with GH45 subfamily A, B and C.

The enzymatic hydrolysis of barley beta-glucan, konjac glucomannan and carboxymethyl cellulose by a ß-1,4-D-endoglucanase MeCel45A from blue mussel, Mytilus edulis, which belongs to subfamily B of glycoside hydrolase family 45 (GH45), was compared with GH45 members of subfamilies A (Humicola insolens HiCel45A), B (Trichoderma reesei TrCel45A) and C (Phanerochaete chrysosporium PcCel45A). Furthermore, the crystal structure of MeCel45A is reported. Initial rates and hydrolysis yields were determined by reducing sugar assays and product formation was characterized using NMR spectroscopy. The subfamily B and C enzymes exhibited mannanase activity, whereas the subfamily A member was uniquely able to produce monomeric glucose. All enzymes were confirmed to be inverting glycoside hydrolases. MeCel45A appears to be cold adapted by evolution, as it maintained 70% activity on cellohexaose at 4 °C relative to 30 °C, compared to 35% for TrCel45A. Both enzymes produced cellobiose and cellotetraose from cellohexaose, but TrCel45A additionally produced cellotriose.

Selectivity mechanism of phosphodiesterase isoform inhibitor through in silico investigations.

Understanding the selectivity mechanism of inhibitors towards homology proteins helps to design selective candidates. Phosphodiesterase (PDE) family members act in the degradation of cAMP and cGMP, among which some isoforms such as PDE9A are attracting interest for Alzheimer's disease treatment, while PDE10A is used as target for treating schizophrenia. In this study, computational methods were used to investigate the major features of PDE9A/10A, with the purpose to provide deep understanding of the molecular mechanism of selective inhibition towards these two isoforms. Our result revealed that two conserved residues Gln453 and Phe456 were proven to be crucial for the binding affinity and inhibitory selectivity of PDE9A inhibitors. In addition, the high-affinity PDE9A inhibitors always interact with the conservative hydrophobic pocket as well as Tyr424 and Ala452 of PDE9A, while PDE10A selective inhibitors need to have two hydrophobic groups and two hydrogen bond donors to interact with the conservative Tyr693, Gln726, and Phe729 of PDE10A. This study provides valuable insights into the underlying mechanism of selective inhibition targeting PDE9A and PDE10A, for further search for potent and highly selective PDE9A/10A inhibitors.

An electrostatic duel: subtle differences in the catalytic performance of monoamine oxidase A and B isoenzymes elucidated at the residue level using quantum computations.

The origin of the immense catalytic power of enzymes remains one of the biggest unresolved questions in biochemistry, with electrostatics being one of the main contenders. Herein, we report results that not only confirm that electrostatics is the driving force behind enzyme catalysis, but also that it is capable of tuning subtle differences in the catalytic performance between structurally similar enzymes, as demonstrated using the example of isoenzymes, monoamine oxidases A and B. Using our own computationally efficient multiscale model [A. Prah, et al., ACS Catal., 2019, 9, 1231] we analyzed the rate-limiting step of the reaction between phenylethylamine and both isoenzymes and deduced that the electrostatic environment provided by isoenzyme B has a perceivably higher catalytic influence on all the considered parameters of the reaction (energy barrier, charge transfer, dipole moment, and HOMO-LUMO gap). This is in full agreement with the available experimental kinetic data and with our own simulations of the reaction in question. In-depth analysis of individual amino acid contributions of both isoenzymes to the barrier (based on the interaction between the electric field provided by the enzyme and the dipole moment of the reacting moiety) shows that the majority of the difference between the isoenzymes can be attributed to a small number of sizable differences between the aligned amino acid pairs, whereas in most of the pairs the difference in contribution to the barrier is vanishingly small. These results suggest that electrostatics largely controls the substrate selectivity of enzymes and validates our approach as being capable of discerning fine nuances in the selectivity of structurally related isoenzymes.

Identification of the first highly selective inhibitor of human lactate dehydrogenase B.

Lactate dehydrogenase (LDH) catalyses the conversion of pyruvate to lactate and NADH to NAD+; it has two isoforms, LDHA and LDHB. LDHA is a promising target for cancer therapy, whereas LDHB is necessary for basal autophagy and cancer cell proliferation in oxidative and glycolytic cancer cells. To the best of our knowledge, selective inhibitors for LDHB have not yet been reported. Here, we developed a high-throughput mass spectrometry screening system using an LDHB enzyme assay by detecting NADH and NAD+. As a result, we identified a small-molecule LDHB selective inhibitor AXKO-0046, an indole derivative. This compound exhibited uncompetitive LDHB inhibition (EC50 = 42 nM). X-ray crystallography revealed that AXKO-0046 bound to the potential allosteric site away from the LDHB catalytic active site, suggesting that targeting the tetramerisation interface of the two dimers is critical for the enzymatic activity. AXKO-0046 and its derivatives can be used to validate LDHB-associated pathways in cancer metabolism.

Structural basis of the P4B ATPase lipid flippase activity.

P4 ATPases are lipid flippases that are phylogenetically grouped into P4A, P4B and P4C clades. The P4A ATPases are heterodimers composed of a catalytic α-subunit and accessory ß-subunit, and the structures of several heterodimeric flippases have been reported. The S. cerevisiae Neo1 and its orthologs represent the P4B ATPases, which function as monomeric flippases without a ß-subunit. It has been unclear whether monomeric flippases retain the architecture and transport mechanism of the dimeric flippases. Here we report the structure of a P4B ATPase, Neo1, in its E1-ATP, E2P-transition, and E2P states. The structure reveals a conserved architecture as well as highly similar functional intermediate states relative to dimeric flippases. Consistently, structure-guided mutagenesis of residues in the proposed substrate translocation path disrupted Neo1's ability to establish membrane asymmetry. These observations indicate that evolutionarily distant P4 ATPases use a structurally conserved mechanism for substrate transport.

Role of active site arginine residues in substrate recognition by PPM1A.

Reversible protein phosphorylation is a key mechanism for regulating numerous cellular events. The metal-dependent protein phosphatases (PPM) are a family of Ser/Thr phosphatases, which uniquely recognize their substrate as a monomeric enzyme. In the case of PPM1A, it has the capacity to dephosphorylate a variety of substrates containing different sequences, but it is not yet fully understood how it recognizes its substrates. Here we analyzed the role of Arg33 and Arg186, two residues near the active site, on the dephosphorylation activity of PPM1A. The results showed that both Arg residues were critical for enzymatic activity and docking-model analysis revealed that Arg186 is positioned to interact with the substrate phosphate group. In addition, our results suggest that which Arg residue plays a more significant role in the catalysis depends directly on the substrate.

Identification of Phosphorylated Calpain 3 in Rat Brain Mitochondria under mPTP Opening.

The protein phosphorylation of the membrane-bound mitochondrial proteins has become of interest from the point of view of its regulatory role of the function of the respiratory chain, opening of the mitochondrial permeability transition pore (mPTP), and initiation of apoptosis. Earlier, we noticed that upon phosphorylation of proteins in some proteins, the degree of their phosphorylation increases with the opening of mPTP. Two isoforms of myelin basic protein and cyclic nucleotide phosphodiesterase were identified in rat brain non-synaptic mitochondria and it was concluded that they are involved in mPTP regulation. In the present study, using the mass spectrometry method, the phosphorylated protein was identified as Calpain 3 in rat brain non-synaptic mitochondria. In the present study, the phosphoprotein Calpain-3 (p94) (CAPN3) was identified in the rat brain mitochondria as a phosphorylated truncated form of p60-62 kDa by two-dimensional electrophoresis and mass spectrometry. We showed that the calpain inhibitor, calpeptin, was able to suppress the Ca2+ efflux from mitochondria, preventing the opening of mPTP. It was found that phosphorylated truncated CALP3 with a molecular weight of 60-62 contains p-Tyr, which indicates the possible involvement of protein tyrosine phosphatase in this process.

Molecular characterization and expressional analysis of two poly (ADP-ribose) polymerase (PARP) domain-containing Gig2 isoforms in rockfish (Sebastes schlegelii) and their antiviral activity against viral hemorrhagic septicemia virus.

Remedies toward sustainable aquaculture rely upon research that unveils the molecular mechanisms behind host immunity and their interactions with pathogens. Antiviral defense is a major innate immune response in fish. The antiviral protein GCHV-induced gene-2 (Gig2), a member of the interferon-stimulated gene (ISG), was identified and characterized from rockfish (Sebastes schlegelii). Gig2 exists in two isoforms, namely, SsGig2-I1 and SsGig2-I2, in rockfish with lengths of 163 and 223 bp, respectively. Bioinformatic analysis indicated the availability of poly (ADP-ribose) polymerase domain in both proteins, and 51.3% identity and 71.3% similarity between both isoforms were observed. The basal expression pattern revealed the highest tissue-specific expression in rockfish gills for both isoforms. The immune challenge experiment disclosed a distinctive and strong expression of each transcript in the presence of poly I:C. Both isoforms are localized in the endoplasmic reticulum. Interferon (IFN) pathway gene analysis revealed no significant upregulation of IFN related genes. Viral hemorrhagic septicemia virus (VHSV) gene expression analysis revealed strong downregulation of viral transcripts after 48 h of infection in the presence of Gig2 isoforms. Collectively, these results indicate the protective role of Gig2 in rockfish against VHSV infection and help broaden our understanding of the innate immunity of fish.

Active site characterization and activity of the human aspartyl (asparaginyl) ß-hydroxylase.

Human aspartyl/asparaginyl beta-hydroxylase (HAAH) is a member of the superfamily of nonheme Fe2+/α-ketoglutarate (αKG) dependent oxygenase enzymes with a noncanonical active site. HAAH hydroxylates epidermal growth factor (EGF) like domains to form the ß-hydroxylated product from substrate asparagine or aspartic acid and has been suggested to have a negative impact in a variety of cancers. In addition to iron, HAAH also binds divalent calcium, although the role of the latter is not understood. Herein, the metal binding chemistry and influence on enzyme stability and activity have been evaluated by a combined biochemical and biophysical approach. Metal binding parameters for the HAAH active site were determined by use of isothermal titration calorimetry, demonstrating a high-affinity regulatory binding site for Ca2+ in the catalytic domain in addition to the catalytic Fe2+ cofactor. We have analyzed various active site derivatives, utilizing LC-MS and a new HPLC technique to determine the role of metal binding and the second coordination sphere in enzyme activity, discovering a previously unreported residue as vital for HAAH turnover. This analysis of the in vitro biochemical function of HAAH furthers the understanding of its importance to cellular biochemistry and metabolic pathways.

Synthesis of 3-O-Sulfated Heparan Sulfate Oligosaccharides Using 3-O-Sulfotransferase Isoform 4.

Heparan sulfate (HS) 3-O-sulfotransferase isoform 4 (3-OST-4) is a specialized carbohydrate sulfotransferase participating in the biosynthesis of heparan sulfate. Here, we report the expression and purification of the recombinant 3-OST-4 enzyme and use it for the synthesis of a library of 3-O-sulfated hexasaccharides and 3-O-sulfated octasaccharides. The unique structural feature of the library is that each oligosaccharide contains a disaccharide domain with a 2-O-sulfated glucuronic acid (GlcA2S) and 3-O-sulfated glucosamine (GlcNS3S). By rearranging the order of the enzymatic modification steps, we demonstrate the synthesis of oligosaccharides with different saccharide sequences. The structural characterization was completed by electrospray ionization mass spectrometry and NMR. These 3-O-sulfated oligosaccharides show weak to very weak anti-Factor Xa activity, a measurement of anticoagulant activity. We discovered that HSoligo 7 (HS oligosaccharide 7), a 3-O-sulfated octasaccharide, binds to high mobility group box 1 protein (HMGB1) and tau protein, both believed to be involved in the process of inflammation. Access to the recombinant 3-OST-4 expands the capability of the chemoenzymatic method to synthesize novel 3-O-sulfated oligosaccharides. The oligosaccharides will become valuable reagents to probe the biological functions of 3-O-sulfated HS and to develop HS-based therapeutic agents.