The crystal and solution studies of glucosamine-6-phosphate synthase from Candida albicans

Glucosamine 6-phosphate (GlcN-6-P) synthase is an ubiquitous enzyme that catalyses the first committed step in the reaction pathway that leads to formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), a precursor of macromolecules that contain amino sugars. Despite sequence similarities, the enzyme in eukaryotes is tetrameric, whereas in prokaryotes it is a dimer. The activity of eukaryotic GlcN-6-P synthase (known as Gfa1p) is regulated by feedback inhibition by UDP-GlcNAc, the end product of the reaction pathway, whereas in prokaryotes the GlcN-6-P synthase (known as GlmS) is not regulated at the post-translational level. In bacteria and fungi the enzyme is essential for cell wall synthesis. In human the enzyme is a mediator of insulin resistance. For these reasons, Gfa1p is a target in anti-fungal chemotherapy and in therapeutics for type-2 diabetes. The crystal structure of the Gfa1p isomerase domain from Candida albicans has been analysed in complex with the allosteric inhibitor UDP-GlcNAc and in the presence of glucose 6-phosphate, fructose 6-phosphate and an analogue of the reaction intermediate, 2-amino-2-deoxy-d-mannitol 6-phosphate (ADMP). A solution structure of the native Gfa1p has been deduced using small-angle X-ray scattering (SAXS). The tetrameric Gfa1p can be described as a dimer of dimers, with each half similar to the related enzyme from Escherichia coli. The core of the protein consists of the isomerase domains. UDP-GlcNAc binds, together with a metal cation, in a well-defined pocket on the surface of the isomerase domain. The residues responsible for tetramerisation and for binding UDP-GlcNAc are conserved only among eukaryotic sequences. Comparison with the previously studied GlmS from E. coli reveals differences as well as similarities in the isomerase active site. This study of Gfa1p focuses on the features that distinguish it from the prokaryotic homologue in terms of quaternary structure, control of the enzymatic activity and details of the isomerase active site.     

Glucosamine-6-phosphate synthase--the multi-facets enzyme

L-Glutamine: D-fructose-6-phosphate amidotransferase, known under trivial name of glucosamine-6-phosphate synthase, as the only member of the amidotransferase subfamily of enzymes, does not display any ammonia-dependent activity. This enzyme, catalysing the first committed step in a pathway leading to the eventual formation of uridine 5'-diphospho-N-acetyl-D-glucosamine (UDP-GlcNAc), is an important point of metabolic control in biosynthesis of amino sugar-containing macromolecules. The molecular mechanism of reaction catalysed by GlcN-6-P synthase is complex and involves both amino transfer and sugar isomerisation. Substantial alterations to the enzyme structure and properties have been detected in different neoplastic tissues. GlcN-6-P synthase is inflicted in phenomenon of hexosamine-induced insulin resistance in diabetes. Finally, this enzyme has been proposed as a promising target in antifungal chemotherapy. Most of these issues, especially their molecular aspects, have been extensively studied in recent years. This article provides a comprehensive overview of the present knowledge on this multi-facets enzyme.     

Kinetic characterization of human glutamine-fructose-6-phosphate amidotransferase I: potent feedback inhibition by glucosamine 6-phosphate

Glutamine-fructose-6-phosphate amidotransferase (GFAT) catalyzes the first committed step in the pathway for biosynthesis of hexosamines in mammals. A member of the N-terminal nucleophile class of amidotransferases, GFAT transfers the amino group from the L-glutamine amide to D-fructose 6-phosphate, producing glutamic acid and glucosamine 6-phosphate. The kinetic constants reported previously for mammalian GFAT implicate a relatively low affinity for the acceptor substrate, fructose 6-phosphate (Fru-6-P, K(m) 0.2-1 mm). Utilizing a new sensitive assay that measures the production of glucosamine 6-phosphate (GlcN-6-P), purified recombinant human GFAT1 (hGFAT1) exhibited a K(m) for Fru-6-P of 7 microm, and was highly sensitive to product inhibition by GlcN-6-P. In a second assay method that measures the stimulation of glutaminase activity, a K(d) of 2 microm was measured for Fru-6-P binding to hGFAT1. Further, we report that the product, GlcN-6-P, is a potent competitive inhibitor for the Fru-6-P site, with a K(i) measured of 6 microm. Unlike other members of the amidotransferase family, where glutamate production is loosely coupled to amide transfer, we have demonstrated that hGFAT1 production of glutamate and GlcN-6-P are strictly coupled in the absence of inhibitors. Similar to other amidotransferases, competitive inhibitors that bind at the synthase site may inhibit the synthase activity without inhibiting the glutaminase activity at the hydrolase domain. GlcN-6-P, for example, inhibited the transfer reaction while fully activating the glutaminase activity at the hydrolase domain. Inhibition of hGFAT1 by the end product of the pathway, UDP-GlcNAc, was competitive with a K(i) of 4 microm. These data suggest that hGFAT1 is fully active at physiological levels of Fru-6-P and may be regulated by its product GlcN-6-P in addition to the pathway end product, UDP-GlcNAc.     

Dynamic actions of glucose and glucosamine on hexosamine biosynthesis in isolated adipocytes: differential effects on glucosamine 6-phosphate, UDP-N-acetylglucosamine, and ATP levels

Glucose and glucosamine (GlcN) cause insulin resistance over several hours by increasing metabolite flux through the hexosamine biosynthesis pathway (HBP). To elucidate the early events underlying glucose-induced desensitization, we treated isolated adipocytes with either glucose or GlcN and then measured intracellular levels of glucose-6-P (G-6-P), GlcN-6-P, UDP-Glc-NAc, and ATP. Glucose treatment rapidly increased G-6-P levels (t((1/2)) < 1 min), which plateaued by 15 min and remained elevated for up to 4 h (glucose ED(50) = 4mm). In glucose-treated cells, GlcN-6-P was undetectable; however, GlcN treatment (2 mm) caused a rapid and massive accumulation of GlcN-6-P. Levels increased by 5 min ( approximately 400 nmol/g) and continued to rise over 2 h (t((1/2)) approximately 20 min) before reaching a plateau at >1,400 nmol/g (ED(50) = 900 microm). Thus, at high GlcN concentrations, unrestricted flux into the HBP greatly exceeds the biosynthetic capacity of the pathway leading to a rapid buildup of GlcN-6-P. The GlcN-induced rise in GlcN-6-P levels was correlated with ATP depletion, suggesting that ATP loss is caused by phosphate sequestration (with the formation of GlcN-6-P) or the energy demands of phosphorylation. As expected, GlcN and glucose increased UDP-GlcNAc levels (t((1/2)) approximately 14-18 min), but greater levels were obtained with GlcN (4-5-fold for GlcN, 2-fold for glucose). Importantly, we found that low doses of GlcN (<250 microm, ED(50) = 80 microm) could markedly elevate UDP-GlcNAc levels without increasing GlcN-6-P levels or depleting ATP levels. These studies on the dynamic actions of glucose and GlcN on hexosamine levels should be useful in exploring the functional role of the HBP and in avoiding the potential pitfalls in the pharmacological use of GlcN.     

Modification of quaternary structure of Candida albicans GlcN-6-P synthase and its desensitization to inhibition by UDP-GlcNAc by site-directed mutagenesis

Site-directed mutagenesis of the CaGFA1 gene encoding glucosamine-6-phosphate synthase from Candida albicans was performed. Desensitization of the enzyme to inhibition by UDPGlcNAc was achieved upon T487I and H492F substitutions at the UDP-GlcNAc binding site, exchange of D524, S525 and S527 for Ala at the dimer:dimer interface and construction of the tail-lock array (L434R and L460A) at the C-tail region. The first two sets if mutageneses but not the last one resulted in conversion of the tetrameric enzyme into its dimeric form. Evidence for links and communication between the UDP-GlcNAc binding site and the dimer-dimer contact areas are presented. The CaGfa1-T487IH492F and CaGfa1-KHSH-D524AS525AS527A muteins are the first examples of the successful conversion of eukaryotic GlcN-6-P synthase into its prokaryotic-like version upon rational site-directed mutagenesis.     

Structural analogues of reactive intermediates as inhibitors of glucosamine-6-phosphate synthase and phosphoglucose isomerase

The active centers of phosphoglucose isomerase (PGI) and the hexose phosphate isomerase domain (HPI) of glucosamine-6-P (GlcN-6-P) synthase demonstrate apparent similarity in spatial arrangement of critical amino acid residues, except Arg272 of the former and Lys603 and Lys485 of the latter. Ten derivatives of d-hexitol-6-P, 5-phosphoarabinoate, or 6-phosphogluconate, structural analogues of putative cis-enolamine or cis-enolate intermediates, were tested as inhibitors of fungal GlcN-6-P synthase and PGI. None of the investigated compounds demonstrated equally high inhibitory potential against both enzymes. 2-Amino-2-deoxy-D-mannitol 6-P was found to be the strongest GlcN-6-P synthase inhibitor in the series, with an inhibition constant equal to 9.0 (+/-1.0) x 10(-6)M. On the contrary, 5-phosphoarabinoate (5PA) exhibited specificity for PGI, with K(i)=2.2 (+/-0.1) x 10(-6) M. N-acetylation substantially lowered the GlcN-6-P synthase inhibitory potential of 2-amino-2-deoxy-D-glucitol-6-P but strongly enhanced inhibitory potential of this compound towards PGI. Molecular modeling studies revealed that interactions of the C1-C2 part of transition state analogue inhibitors with the respective areas demonstrating different distribution of molecular electrostatic potential (MEP) inside HPI and PGI active centers determined enzyme:ligand affinity. In Escherichia coli HPI, a patch of the negative potential created by Glu488 aided by Val399, supposed to stabilize a putative positively charged intermediate, especially attracts ligands containing 2-amino function. The Arg272, Lys210, and Gly271 peptide bond nitrogen system, present in the corresponding space of rabbit PGI, creates an area of positive MEP, stabilizing cis-enolate intermediate and attracting its structural mimics, such as 5PA.     

Design and synthesis of novel cell wall inhibitors of Mycobacterium tuberculosis GlmM and GlmU

GlmM and GlmU are key enzymes in the biosynthesis of UDP-N-acetyl-d-glucosamine (UDP-GlcNAc), an essential precursor of peptidoglycan and the rhamnose–GlcNAc linker region in the mycobacterial cell wall. These enzymes are involved in the conversion of two important precursors of UDP-GlcNAc, glucosamine-6-phosphate (GlcN-6-P) and glucosamine-1-phosphate (GlcN-1-P). GlmM converts GlcN-6-P to GlcN-1-P, GlmU is a bifunctional enzyme, whereby GlmU converts GlcN-1-P to GlcNAc-1-P and then catalyzes the formation of UDP-GlcNAc from GlcNAc-1-P and uridine triphosphate. In the present study, methyl 2-amino-2-deoxyl-α-d-glucopyranoside 6-phosphate (1α), methyl 2-amino-2-deoxyl-β-d-glucopyranoside 6-phosphate (1β), two analogs of GlcN-6-P, were synthesized as GlmM inhibitors; 2-azido-2-deoxy-α-d-glucopyranosyl phosphate (2) and 2-amino-2,3-dideoxy-3-fluoro-α-d-glucopyranosyl phosphate (3), analogs of GlcN-1-P, were synthesized firstly as GlmU inhibitors. Compounds 1α, 1β, 2, and 3 as possible inhibitors of mycobacterial GlmM and GlmU are reported herein. Compound 3 showed promising inhibitory activities against GlmU, whereas 1α, 1β and 2 were inactive against GlmM and GlmU even at high concentrations.     

Glucosamine-6-phosphate synthase, a novel target for antifungal agents. Molecular modelling studies in drug design

Fungal infections are a growing problem in contemporary medicine, yet only a few antifungal agents are used in clinical practice. In our laboratory we proposed the enzyme L-glutamine: D-fructose-6-phosphate amidotransferase (EC 2.6.1.16) as a new target for antifungals. The structure of this enzyme consists of two domains, N-terminal and C-terminal ones, catalysing glutamine hydrolysis and sugar-phosphate isomerisation, respectively. In our laboratory a series of potent selective inhibitors of GlcN-6-P synthase have been designed and synthesised. One group of these compounds, including the most studied N3-(4-methoxyfumaroyl)-l-2,3-diaminopropanoic acid (FMDP), behave like glutamine analogs acting as active-site-directed inactivators, blocking the N-terminal, glutamine-binding domain of the enzyme. The second group of GlcN-6-P synthase inhibitors mimic the transition state of the reaction taking place in the C-terminal sugar isomerising domain. Surprisingly, in spite of the fact that glutamine is the source of nitrogen for a number of enzymes it turned out that the glutamine analogue FMDP and its derivatives are selective against GlcN-6-P synthase and they do not block other enzymes, even belonging to the same family of glutamine amidotransferases. Our molecular modelling studies of this phenomenon revealed that even within the family of related enzymes substantial differences may exist in the geometry of the active site. In the case of the glutamine amidotransferase family the glutamine binding site of GlcN-6-P synthase fits a different region of the glutamine conformational space than other amidotransferases. Detailed analysis of the interaction pattern for the best known, so far, inhibitor of the sugar isomerising domain, namely 2-amino-2-deoxy-D-glucitol-6-phosphate (ADGP), allowed us to suggest changes in the structure of the inhibitor that should improve the interaction pattern. The novel ligand was designed and synthesised. Biological experiments confirmed our predictions. The new compound named ADMP is a much better inhibitor of glucosamine-6-phosphate synthase than ADGP.     

Structure of the E. coli bifunctional GlmU acetyltransferase active site with substrates and products

The biosynthesis of UDP-GlcNAc in bacteria is carried out by GlmU, an essential bifunctional uridyltransferase that catalyzes the CoA-dependent acetylation of GlcN-1-PO(4) to form GlcNAc-1-PO(4) and its subsequent condensation with UTP to form pyrophosphate and UDP-GlcNAc. As a metabolite, UDP-GlcNAc is situated at a branch point leading to the biosynthesis of lipopolysaccharide and peptidoglycan. Consequently, GlmU is regarded as an important target for potential antibacterial agents. The crystal structure of the Escherichia coli GlmU acetyltransferase active site has been determined in complexes with acetyl-CoA, CoA/GlcN-1-PO(4), and desulpho-CoA/GlcNAc-1-PO(4). These structures reveal the enzyme groups responsible for binding the substrates. A superposition of these complex structures suggests that the 2-amino group of GlcN-1-PO(4) is positioned in proximity to the acetyl-CoA to facilitate direct attack on its thioester by a ternary complex mechanism.     

Evaluation of synthase and hemisynthase activities of glucosamine-6-phosphate synthase by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry

Glucosamine-6-phosphate synthase (GlmS, EC 2.6.1.16) catalyzes the first and rate-limiting step in the hexosamine biosynthetic pathway, leading to the synthesis of uridine-5′-diphospho-N-acetyl-d-glucosamine, the major building block for the edification of peptidoglycan in bacteria, chitin in fungi, and glycoproteins in mammals. This bisubstrate enzyme converts d-fructose-6-phosphate (Fru-6P) and l-glutamine (Gln) into d-glucosamine-6-phosphate (GlcN-6P) and l-glutamate (Glu), respectively. We previously demonstrated that matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) allows determination of the kinetic parameters of the synthase activity. We propose here to refine the experimental protocol to quantify Glu and GlcN-6P, allowing determination of both hemisynthase and synthase parameters from a single assay kinetic experiment, while avoiding interferences encountered in other assays. It is the first time that MALDI-MS is used to survey the activity of a bisubstrate enzyme.     

Inhibitors of glucosamine-6-phosphate synthase as potential antimicrobials or antidiabetics – synthesis and properties

Glucosamine-6-phosphate synthase (GlcN-6-P synthase) is known as a promising target for antimicrobial agents and antidiabetics. Several compounds of natural or synthetic origin have been identified as inhibitors of this enzyme. This set comprises highly selective l-glutamine, amino sugar phosphate or transition state intermediate cis-enolamine analogues. Relatively low antimicrobial activity of these inhibitors, poorly penetrating microbial cell membranes, has been improved using the pro-drug approach. On the other hand, a number of heterocyclic and polycyclic compounds demonstrating antimicrobial activity have been presented as putative inhibitors of the enzyme, based on the results of molecular docking to GlcN-6-P synthase matrix. The most active compounds of this group could be considered promising leads for development of novel antimicrobial drugs or antidiabetics, provided their selective toxicity is confirmed.     

Glucosamine-induced activation of glycogen biosynthesis in isolated adipocytes. Evidence for a rapid allosteric control mechanism within the hexosamine biosynthesis pathway

Enhanced flux through the hexosamine biosynthesis pathway (HBP) induces insulin resistance and facilitates lipid storage through the up-regulation of enzyme mRNA levels. Both actions occur over several hours and require gene expression. We now identify a regulatory arm of the HBP that involves rapid allosteric activation of glycogen synthase (GS) and stimulation of glycogen biosynthesis (GBS). When insulin-pretreated adipocytes were exposed to 2 mM GlcN, incorporation of [14C]glucose into glycogen doubled by 10 min (t(1/2) of <5 min), whereas UDP-glucose levels were concomitantly decreased during this time (t(1/2) of 1.4 min; >90% depletion). Stimulation of GBS and depletion of UDP-glucose both correlated with an early and rapid rise in the levels of glucosamine-6-phosphate (GlcN-6-P), a known activator of GS. The lowering of GlcN-6-P levels by removing extracellular GlcN (>80% reduction by 45 min) was accompanied by the restoration of UDP-glucose levels. Prolonged GlcN treatment (20 min to 2 h) inhibited GBS, which corresponded to a massive intracellular accumulation of GlcN-6-P (t(1/2) of approximately 32 min; >1,400 nmol/g). From these data, we conclude the following. 1) GlcN treatment elevated intracellular GlcN-6-P levels within minutes, resulting in allosteric activation of GS, stimulation of GBS, and a reduction in steady-state levels of UDP-glucose due to increased precursor utilization. 2) Prolonged treatment with high concentrations of GlcN caused massive accumulation of GlcN-6-P that adversely affected cellular metabolism and reduced GBS. 3) The biphasic actions of GlcN on GBS may explain many of the discrepant reports on the role of the HBP in glycogen metabolism.     

Functional domains and interdomain communication in Candida albicans glucosamine-6-phosphate synthase

Functional and structural properties of several truncated or mutated variants of Candida albicans Gfa1p (glucosamine-6-phosphate synthase) were compared with those of the wild-type enzyme. Fragments encompassing residues 1-345 and 346-712 of Gfa1p, expressed heterogeneously in bacterial host as His6 fusions, were identified as the functional GAH (glutamine amidehydrolysing) and ISOM (hexose phosphate-isomerizing) domains respectively. It was found that the native GAH domain is monomeric, whereas the native ISOM domain forms tetramers, as does the whole enzyme. Spectrofluorimetric and kinetic studies of the isolated domains, the Delta218-283Gfa1p mutein and the wild-type enzyme revealed that the binding site for the feedback inhibitor, uridine 5'-diphospho-N-acetyl-D-glucosamine, is located in the ISOM domain. Inhibitor binding affects amidohydrolysing activity of the GAH domain and, as a consequence, the GlcN-6-P (D-glucosamine-6-phosphate)-synthetic activity of the whole enzyme. The fragment containing residues 218-283 is neither involved in ligand binding nor in protein oligomerization. Comparison of the catalytic activities of Gfa1p(V711F), Delta709-712Gfa1p, Gfa1p(W97F) and Gfa1p(W97G) with those of the native Gfa1p and the isolated domains provided evidence for an intramolecular channel connecting the GAH and ISOM domains of Gfa1p. The channel becomes leaky upon deletion of amino acids 709-712 and in the W97F and W97G mutants. The Trp97 residue was found to function as a molecular gate, opening and closing the channel. The W97G and V711F mutations resulted in an almost complete elimination of the GlcN-6-P-synthetic activity, with the retention of the amidohydrolase and sugar phosphate-isomerizing activities.     

Phosphorylation of glucosamine-6-phosphate synthase is important but not essential for germination and mycelial growth of Candida albicans

A site-directed mutagenesis of the GFA1 gene encoding Candida albicans glucosamine-6-phosphate (GlcN-6-P) synthase afforded its GFA1S208A version. A product of the modified gene, lacking the putative phosphorylation site for protein kinase A (PKA), exhibited all the basic properties identical to those of the wild-type enzyme but was no longer a substrate for PKA. Comparison of the C. albicans Deltagfa1/GFA1 and Deltagfa1/GFA1S208A cells, grown under conditions stimulating yeast-to-mycelia transformation, revealed that the latter demonstrated lower GlcN-6-P synthase specific activity, decreased chitin content and formed much fewer mycelial forms. All these findings, as well as the observed effects of specific inhibitors of protein kinases, suggest that a loss of the possibility of GlcN-6-P synthase phosphorylation by PKA strongly reduces but not completely eliminates the germinative response of C. albicans cells.     

Structural, kinetic and computational investigation of Vitis vinifera DHDPS reveals new insight into the mechanism of lysine-mediated allosteric inhibition

Lysine is one of the most limiting amino acids in plants and its biosynthesis is carefully regulated through inhibition of the first committed step in the pathway catalyzed by dihydrodipicolinate synthase (DHDPS). This is mediated via a feedback mechanism involving the binding of lysine to the allosteric cleft of DHDPS. However, the precise allosteric mechanism is yet to be defined. We present a thorough enzyme kinetic and thermodynamic analysis of lysine inhibition of DHDPS from the common grapevine, Vitis vinifera (Vv). Our studies demonstrate that lysine binding is both tight (relative to bacterial DHDPS orthologs) and cooperative. The crystal structure of the enzyme bound to lysine (2.4 Å) identifies the allosteric binding site and clearly shows a conformational change of several residues within the allosteric and active sites. Molecular dynamics simulations comparing the lysine-bound (PDB ID 4HNN) and lysine free (PDB ID 3TUU) structures show that Tyr132, a key catalytic site residue, undergoes significant rotational motion upon lysine binding. This suggests proton relay through the catalytic triad is attenuated in the presence of lysine. Our study reveals for the first time the structural mechanism for allosteric inhibition of DHDPS from the common grapevine.     

Mechanism of palmityl coenzyme A inhibition of liver glycogen synthase.

Palmityl-CoA inhibits free liver glycogen synthase; the concentration required for half-maximum inhibition is 3 to 4 micrometer. Almost complete inhibition was observed at 50 micrometer. Palmityl-CoA inhibition is associated with dissociation of the tetrameric enzyme into monomers, and binding of palmityl-CoA to the monomers. Glycogen-bound enzyme is also inhibited by palmityl-CoA, resulting in dissociation of the enzyme into monomers and concomitant release of the enzyme from the primer glycogen. Palmityl-CoA inhibition of the enzyme is partially reversed by the glycogen synthase activator, glucose-6-P, whereas sodium lauryl sulfate-inhibited enzyme is not reactivated by glucose-6-P. Sodium lauryl sulfate inhibition results in the dissociation of the tetramer into the monomers. Bovine serum albumin and cyclodextrin can prevent palmityl-CoA inhibition only when they are added prior to palmityl-CoA addition. The possible physiological role of palmityl-CoA in glucose homeostasis is discussed.     

Phosphorylation-dependent regulation of amidotransferase during the development of Blastocladiella emersonii

The enzyme amidotransferase [2-amino-2-deoxy-D-glucose-6-phosphate ketol isomerase (amino-transferring); EC 2.6.1.16] catalyzes the first step in the hexosamine biosynthetic pathway. In Blastocladiella emersonii the sensitivity of the enzyme to the inhibitor uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) is developmentally regulated. The inhibitable form of amidotransferase activity present in the zoospore is converted to a noninhibitable form during germination. The latter form is present throughout the growth phase and sensitivity to UDP-GlcNAc gradually returns to the zoospore level during sporulation [C.P. Selitrennikoff, N.E. Dalley, and D.R. Sonneborn (1980) Proc. Natl. Acad. Sci. USA 77, 5998-6002]. The following evidence suggests that a phosphorylation/dephosphorylation mechanism underlies this interconversion: (i) Both the vegetative and zoospore enzymes have the same molecular weight of 140,000, but the vegetative enzyme elutes significantly earlier on a DEAE-cellulose column than does the zoospore enzyme. (ii) The increased sensitivity to UDP-GlcNAc occurring in vivo and in vitro correlates with increased phosphorylation of a polypeptide of apparent Mr 76,000. This component copurifies with amidotransferase activity through ion-exchange chromatography and sucrose density gradient centrifugation. (iii) Desensitization and concurrent dephosphorylation of sensitive amidotransferase can be observed in vitro after treatment with a partially purified magnesium-dependent phosphoprotein phosphatase from zoospores.     

Kinetic properties of <Emphasis Type="Italic">Mycobacterium tuberculosis</Emphasis> bifunctional GlmU

The UDP-N-acetylglucosamine (UDP-GlcNAc) is present as one of the glycosyl donors for disaccharide linker (d-N-GlcNAc-l-rhamnose) and the precursor of peptidoglycan in mycobacteria. The bifunctional enzyme GlmU involves in the last two sequential steps of UDP-GlcNAc synthetic pathway. Glucosamine-1-phosphate acetyltransferase catalyzes the formation of N-acetylglucosamine-1-phosphate (GlcNAc-1-P) from glucosamine-1-phosphate (GlcN-1-P) and acetyl coenzyme A (Acetyl CoA), and N-acetylglucosamine-1-phosphate uridyltransferase catalyzes the synthesis of UDP-GlcNAc from GlcNAc-1-P and UTP. The previous studies demonstrating the essentiality of GlmU to mycobacterial survival supported GlmU as a novel and potential target for TB drugs. In this work, two accurate and simple colorimetric assays based on 96-well microtiter plate were developed to measure the kinetic properties of bifunctional GlmU including initial velocity, optimal temperature, optimal pH, the effect of Mg²⁺, and the kinetic parameters. Both of the colorimetric assays for bifunctional GlmU enzyme activities and the kinetic properties will facilitate high-throughput screening of GlmU inhibitors.     

Theoretical evaluation and improvement on the potency of the rhodanine-based inhibitors for human serotonin N-acetyltransferase

Human serotonin N-acetyltransferase (hAANAT), included in the melatonin biosynthesis, plays a pivotal role in the regulation of the biological clock and the daily rhythm. In this research, a reliable model of hAANAT was first constructed by the homology modelling method. Then the inhibition mode of two representative rhodanine-based inhibitors was explored by molecular dynamics simulations and energy analyses. The results show that the inhibitor class could share a similar inhibition mechanism in which the carboxyl moiety is positioned in the Ac-CoA binding region while the other end spans the serotonin binding pocket. The interaction between the inhibitor's carboxyl and the enzyme seems to be more important according to the decomposition of binding free energy. Based on the proposed inhibition mode, the inhibitor's improvement was carried out to obtain a more potent compound. The newly designed inhibitor, with the larger binding free energy, exhibits the stronger interaction with the related residues of the enzyme by the added chemical groups. This work will shed light on the inhibition mechanism of the rhodanine-based inhibitors and promote the development of a new drug targeting hAANAT.     

Phosphate inhibition of spinach leaf sucrose phosphate synthase as affected by glucose-6-phosphate and phosphoglucoisomerase

The inhibition patterns of inorganic phosphate (Pi) on sucrose phosphate synthase activity in the presence and absence of the allosteric activator glucose-6-P was studied, as well as the effects of phosphoglucoisomerase on fructose-6-P saturation kinetics with and without Pi. In the presence of 5 millimolar glucose-6-P, Pi was a partial competitive inhibitor with respect to both substrates, fructose-6-P and uridine diphosphate glucose. In the absence of glucose-6-P, the inhibition patterns were more complex, apparently because of the interaction of Pi at the activation site as well as the catalytic site. In addition, substrate activation by uridine diphosphate glucose was observed in the absence of effectors. The results suggested that Pi antagonizes glucose-6-P activation of sucrose phosphate synthase by competing with the activator for binding to the modifier site.

The fructose-6-P saturation kinetics were hyperbolic in the absence of phosphoglucoisomerase activity, but became sigmoidal by the addition of excess phosphoglucoisomerase. The sigmoidicity persisted in the presence of Pi, but sucrose phosphate synthase activity was decreased. The apparent sigmoidal response may represent the physiological response of sucrose phosphate synthase to a change in hexose-P concentration because sucrose phosphate synthase operates in the cytosol in the presence of high activities of phosphoglucoisomerase. Thus, the enzymic production of an activator from a substrate represents a unique mechanism for generating sigmoidal enzyme kinetics.