Electrostatics,allostery, and activity of the yeast chorismate mutase

The predicted active site of chorismate mutase of baker's yeast Saccharomyces cerevisiae has been studied by continuum electrostatics, molecular surface/volume calculations, and molecular modeling. Our study shows that despite being subject to an allosteric transition, the enzyme's active-site pocket neither decreased in volume nor deformed significantly in shape between the active R state and the inactive T state. We find that the polar atmosphere in the pocket is responsible for the enzyme's affinity. A single amino acid, Glu23, can adequately account for the atmospheric variation. This residue swings into the active-site pocket from the R state to the T state. In the R state, Glu23 on helix H2 doubly pairs with Arg204 and Lys208 of H11, which is packed against H2. In the T state, a slide occurs between H11 and H2 such that Glu23 can no longer interact with Lys208 and competes with Asp24 for interacting with Arg204. Consequently, Glu23 is found in the T state to couple with Arg157, an active-site residue critical to substrate binding. The tandem sliding of H11 in both monomers profoundly changes the interactions in the dimer interface. The loop between H11 and H12 demonstrates the largest conformational change. Hence, we establish a connection between the allosteric transition and the activity of the enzyme. The conformational change in the transition is suggested to propagate into the active-site pocket via a series of polar interactions that result in polarity reversal in the active-site pocket, which regulates the enzyme's activity. Proteins 31:445–452, 1998. © 1998 Wiley-Liss, Inc.     

Down-regulation of UDP-arabinopyranose mutase reduces the proportion of arabinofuranose present in rice cell walls

Arabinoxylans may account for up to 25% of the mass of grass cell walls. The interactions of these polysaccharides with themselves and with cellulose and lignin is believed to affect the walls physical properties and increase the walls resistance to biochemical conversion to fermentable sugars. Arabinoxylans have a backbone composed of 1,4-linked β-d-xylosyl residues, some of which are substituted at O-2 or O-3 with single arabinofuranosyl (Araf) residues. The Araf residues are likely transferred from UDP-Araf to the xylan backbone by arabinofuranosyltransferases. UDP-Araf is itself formed from UDP-arabinopyranose (UDP-Arap) by UDP-arabinopyranose mutase (UAM). In this study, RNA interference (RNAi) was used to suppress UAM expression in rice plants and thereby reduce the amounts of UDP-Araf available for cell wall synthesis. Several of the transgenic plants had reduced proportions of Araf in their walls together with a decrease in the extent of substitution of the xylan backbone, and a reduction of between 25% and 80% in ferulic acid and p-coumaric acid contents of the cell walls. Those transgenic plants with >25% reduction in the amounts of Araf were dwarfed and infertile.     

The synthesis of pABA: Coupling between the glutamine amidotransferase and aminodeoxychorismate synthase domains of the bifunctional aminodeoxychorismate synthase from Arabidopsis thaliana

Aminodeoxychorismate (ADC) synthase in plants is a bifunctional enzyme containing glutamine amidotransferase (GAT) and ADC synthase (ADCS) domains. The GAT domain releases NH₃ from glutamine and the ADCS domain uses NH₃ to aminate chorismate. This enzyme is involved in folate (vitamin B9) biosynthesis. We produced a stable recombinant GAT–ADCS from Arabidopsis. Its kinetic properties were characterized, and activities and coupling of the two domains assessed. Both domains could operate independently, but not at their optimal capacities. When coupled, the activity of one domain modified the catalytic properties of the other. The GAT activity increased in the presence of chorismate, an activation process that probably involved conformational changes. The ADCS catalytic efficiency was 10⁴ fold higher with glutamine than with NH₄Cl, indicating that NH₃ released from glutamine and used for ADC synthesis did not equilibrate with the external medium. We observed that the GAT activity was always higher than that of ADCS, the excess of NH₃ being released in the external medium. In addition, we observed that ADC accumulation retro-inhibited ADCS activity. Altogether, these results indicate that channeling of NH₃ between the two domains and/or amination of chorismate are the limiting step of the whole process, and that ADC cannot accumulate.     

"Muscle to meat" molecular events and technological transformations: the proteomics insight

Cellular death is characterized by a complex pattern of molecular events that depend on cell type. Specifically, muscle cells first undergo rigor mortis due to ATP depletion, and later, on the time scale of days, muscle fiber degradation due to proteolytic enzyme activity. In the present review, we will refer to proteomic investigations on the post-mortem evolution of the protein patterns of animal muscle cells. These studies, carried out with the application of either bottom-up or top-down methods, are relevant for understanding the biochemical reactions that i) convert muscle to meat, ii) are associated with meat aging and iii) impact on meat tenderness, a feature of significant commercial value. We also report on the proteomic investigations that have been made to analyze the transformation of meat in industrial processes. These studies are primarily aimed at identifying protein patterns and/or individual proteins diagnostic of the quality of the final product.     

When Inhibitors Do Not Inhibit: Critical Evaluation of Rational Drug Design Targeting Chorismate Mutase from Mycobacterium tuberculosis

Tuberculosis (TB) is a devastating disease that claims millions of lives every year. Hindered access or non‐compliance to medication, especially in developing countries, led to drug resistance, further aggravating the situation. With current standard therapies in use for over 50 years and only few new candidates in clinical trials, there is an urgent call for new TB drugs. A powerful tool for the development of new medication is structure‐guided design, combined with virtual screening or docking studies. Here, we report the results of a drug‐design project, which we based on a publication that claimed the structure‐guided discovery of several promising and highly active inhibitors targeting the secreted chorismate mutase (*MtCM) from Mycobacterium tuberculosis. We set out to further improve on these compounds and synthesized a series of new derivatives. Thorough evaluation of these molecules in enzymatic assays revealed, to our dismay, that neither the claimed lead compounds, nor any of the synthesized derivatives, show any inhibitory effects against *MtCM.     

Metabolic design of a platform Escherichia coli strain producing various chorismate derivatives

A synthetic metabolic pathway suitable for the production of chorismate derivatives was designed in Escherichia coli. An L-phenylalanine-overproducing E. coli strain was engineered to enhance the availability of phosphoenolpyruvate (PEP), which is a key precursor in the biosynthesis of aromatic compounds in microbes. Two major reactions converting PEP to pyruvate were inactivated. Using this modified E.coli as a base strain, we tested our system by carrying out the production of salicylate, a high-demand aromatic chemical. The titer of salicylate reached 11.5 g/L in batch culture after 48 h cultivation in a 2-liter jar fermentor, and the yield from glucose as the sole carbon source exceeded 40% (mol/mol). In this test case, we found that pyruvate was synthesized primarily via salicylate formation and the reaction converting oxaloacetate to pyruvate. In order to demonstrate the generality of our designed strain, we employed this platform for the production of each of 7 different chorismate derivatives. Each of these industrially important chemicals was successfully produced to levels of 1–3 g/L in test tube-scale culture.     

A plant mutase that interconverts UDP-arabinofuranose and UDP-arabinopyranose

Plant cell walls constitute the bulk of the earth renewable source of energy and are a component in the diet of humans and herbivores. l-Arabinofuranosyl (Araf) residues are a quantifiably important constituent of these walls. Plants use uridine diphosphate (UDP)-l-arabinofuranose (UDP-Araf) to donate Araf residues in the biosynthesis of Araf-containing polysaccharides, proteoglycans, and glycoproteins. However, little is known about the formation of UDP-Araf. We now describe the purification and partial characterization of a rice UDP-arabinopyranose mutase (UAM) that catalyzes the formation of UDP-Araf from UDP-arabinopyranose (UDP-Arap). The reaction is reversible and at thermodynamic equilibrium the pyranose form is favored over the furanose form (90 : 10). Three related proteins that are encoded by rice gene loci Os03g40270, Os04g56520, and Os07g41360 were identified from partial amino acid sequences of UAM. These proteins have >80% sequence identity with polypeptides that are reversibly glycosylated in the presence of UDP-sugars. The rice mutase and two functionally active recombinant mutases were shown to be reversibly glycosylated in the presence of UDP-Glc. The cofactor, flavin-adenine-dinucleotide (FAD), is required for the catalytic activity of UDP-galactose mutases of prokaryotes, fungi, and protozoa. The plant mutases, which do not require a cofactor, must therefore have a different catalytic mechanism. Putative UAM-encoding genes are present in the green algae Chlamydomonas reinhardtii, the moss Physcomitrella patens, the gymnosperm Pinus taeda (loblolly pine), and in numerous dicots and monocots, indicating that UAMs are widespread in green plants.     

Characterization of recombinant UDP-galactopyranose mutase from Aspergillus fumigatus

UDP-galactopyranose mutase (UGM) is a flavin-containing enzyme that catalyzes the conversion of UDP-galactopyranose to UDP-galactofuranose, the precursor of galactofuranose, which is an important cell wall component in Aspergillus fumigatus and other pathogenic microbes. A. fumigatus UGM (AfUGM) was expressed in Escherichia coli and purified to homogeneity. The enzyme was shown to function as a homotetramer by size-exclusion chromatography and to contain ∼50% of the flavin in the active reduced form. A k_cat value of 72 ± 4 s⁻¹ and a K_M value of 110 ± 15 μM were determined with UDP-galactofuranose as substrate. In the oxidized state, AfUGM does not bind UDP-galactopyranose, while UDP and UDP-glucose bind with K_d values of 33 ± 9 μM and 90 ± 30 μM, respectively. Functional and structural differences between the bacterial and eukaryotic UGMs are discussed.     

Separation and characterization of two chorismate-mutase isoenzymes from Nicotiana silvestris

Two isoenzymes of chorismate mutase (EC 5.4.99.5) were isolated and partially purified from leaves of diploid (2n=24) Nicotiana silvestris Speg. et Comes and from isogenic cells in a suspension culture originally established from haploid tissue. An isoenzyme denoted CM-1 (M _r=52,000) accounted for the major fraction of total activity recovered from suspension-cultured cells, while isoenzyme CM-2 (M _r=65,000) represented the major fraction of activity recovered from green leaf tissue. The ratio of isoenzyme levels from these two sources differed more than 20-fold. The subcellular location of isoenzyme CM-1 is known to be in the chloroplasts of green leaves or in proplastids of cultured cells, while isoenzyme CM-2 is located in the cytosol. Both isoenzymes were stable during partial purification, possessed broad pH optima for catalysis between 6.0 and 8.0, and were active without denaturation at temperatures at least as high as 45° C. Thiol reagents were unnecessary for either stability or activity of both isoenzymes. The affinity of isoenzyme CM-2 for substrate (K _m=0.24 mM) was almost an order of magnitude better than that of CM-1. The kinetic behavior of isoenzyme CM-1 was influenced by pH, while that of isoenzyme CM-2 was not. At pH 7.2, hyperbolic substrate-saturation curves (K _m=1.7 mM) were obtained for isoenzyme CM-1. At pH 6.1, however, isoenzyme CM-1 displayed relatively weak positive cooperativity, Hill plots yielding an n value of 1.2 At pH 6.1 the half-saturation ([S]_0.5) value was 2.5 mM.Abbreviations DEAE diethylaminoethyl - M _r molecular weight