A glutamate residue in the catalytic center of the yeast chorismate mutase restricts enzyme activity to acidic conditions

Chorismate mutase acts at the first branchpoint of aromatic amino acid biosynthesis and catalyzes the conversion of chorismate to prephenate. Comparison of the x-ray structures of allosteric chorismate mutase from the yeast Saccharomyces cerevisiae with Escherichia coli chorismate mutase/prephenate dehydratase suggested conserved active sites between both enzymes. We have replaced all critical amino acid residues, Arg-16, Arg-157, Lys-168, Glu-198, Thr-242, and Glu-246, of yeast chorismate mutase by aliphatic amino acid residues. The resulting enzymes exhibit the necessity of these residues for catalytic function and provide evidence of their localization at the active site. Unlike some bacterial enzymes, yeast chorismate mutase has highest activity at acidic pH values. Replacement of Glu-246 in the yeast chorismate mutase by glutamine changes the pH optimum for activity of the enzyme from a narrow to a broad pH range. These data suggest that Glu-246 in the catalytic center must be protonated for maximum catalysis and restricts optimal activity of the enzyme to low pH.

Use of intrinsic binding energy for catalysis by an RNA enzyme

The contribution of several individual ribozyme⋅substrate base pairs to binding and catalysis has been investigated using hammerhead ribozyme substrates that were truncated at their 3′ or 5′ ends. The base pairs at positions 1.1–2.1 and 15.2–16.2, which flank the conserved core, each contribute 104-fold in the chemical step, without affecting substrate binding. In contrast, base pairs distal to the core contribute to substrate binding but have no effect on the chemical step. These results suggest a “fraying model” in which each ribozyme⋅substrate helix can exist in either an unpaired (“open”) state or a helical (“closed”) state, with the closed state required for catalysis. The base pairs directly adjacent to the conserved core contribute to catalysis by allowing the closed state to form. Once the number of base pairs is sufficient to ensure that the closed helical state predominates, additional residues provide stabilization of the helix, and therefore increase binding, but have no further effect on the chemical step. Remarkably, the >5 kcal/mol free energy contribution to catalysis from each of the internal base pairs is considerably greater than the free energy expected for formation of a base pair. It is suggested that this unusually large energetic contribution arises because free energy that is typically lost in constraining residues within a base pair is expressed in the transition state, where it is used for positioning. This extends the concept of “intrinsic binding energy” from protein to RNA enzymes, suggesting that intrinsic binding energy is a fundamental feature of biological catalysis.

The role of the synthetic enzyme GAD65 in the control of neuronal γ-aminobutyric acid release

We have studied GABAergic synaptic transmission in retinal ganglion cells and hippocampal pyramidal cells to determine, at a cellular level, what is the effect of the targeted disruption of the gene encoding the synthetic enzyme GAD65 on the synaptic release of γ-aminobutyric acid (GABA). Neither the size nor the frequency of GABA-mediated spontaneous inhibitory postsynaptic currents (IPSCs) were reduced in retina or hippocampus in GAD65−/− mice. However, the release of GABA during sustained synaptic activation was substantially reduced. In the retina both electrical- and K+-induced increases in IPSC frequency were depressed without a change in IPSC amplitude. In the hippocampus the transient increase in the probability of inhibitory transmitter release associated with posttetanic potentiation was absent in the GAD65−/− mice. These results indicate that during and immediately after sustained stimulation the increase in the probability of transmitter release is not maintained in GAD65−/− mice. Such a finding suggests a decrease in the size or refilling kinetics of the releasable pool of vesicles, and various mechanisms are discussed that could account for such a defect.

Identification of an intramolecular interaction between small regions in type V adenylyl cyclase that influences stimulation of enzyme activity by Gsα

Using the full-length and two engineered soluble forms (C1-C2 and Cla-C2) of type V adenylyl cyclase (ACV), we have investigated the role of an intramolecular interaction in ACV that modulates the ability of the α subunit of the stimulatory GTP-binding protein of AC (Gsα) to stimulate enzyme activity. Concentration–response curves with Gsα suggested the presence of high and low affinity sites on ACV, which interact with the G protein. Activation of enzyme by Gsα interaction at these two sites was most apparent in the C1a-C2 form of ACV, which lacks the C1b region (K572–F683). Yeast two-hybrid data demonstrated that the C1b region interacted with the C2 region and its 64-aa subdomain, C2I. Using peptides corresponding to the C2I region of ACV, we investigated the role of the C1b/C2I interaction on Gsα-mediated stimulation of C1-C2 and full-length ACV. Our data demonstrate that a 10-aa peptide corresponding to L1042–T1051 alters the profile of the activation curves of full-length and C1-C2 forms of ACV by different Gsα concentrations to mimic the activation profile observed with C1a-C2 ACV. The various peptides used in our studies did not alter forskolin-mediated stimulation of full-length and C1-C2 forms of ACV. We conclude that the C1b region of ACV interacts with the 10-aa region (L1042–T1051) in the C2 domain of the enzyme to modulate Gsα-elicited stimulation of activity.

Effects of hydrocarbon contamination on soil microbial community and enzyme activity

Acknowledgment I would like to gratefully acknowledge the government of Saudi Arabia for the scholarship and financial support.

Cleft palate in mice with a targeted mutation in the γ-aminobutyric acid-producing enzyme glutamic acid decarboxylase 67

The functions of neurotransmitters in fetal development are poorly understood. Genetic observations have suggested a role for the inhibitory amino acid neurotransmitter γ-aminobutyric acid (GABA) in the normal development of the mouse palate. Mice homozygous for mutations in the β-3 GABAA receptor subunit develop a cleft secondary palate. GABA, the ligand for this receptor, is synthesized by the enzyme glutamic acid decarboxylase. We have disrupted one of the two mouse Gad genes by gene targeting and also find defects in the formation of the palate. The striking similarity in phenotype between the receptor and ligand mutations clearly demonstrates a role for GABA signaling in normal palate development.

Lactone-ring-cleaving enzyme: Genetic analysis, novel RNA editing, and evolutionary implications

A lactonohydrolase from Fusarium oxysporum AKU 3702 is an enzyme catalyzing the hydrolysis of aldonate lactones to the corresponding aldonic acids. The amino acid sequences of the NH2 terminus and internal peptide fragments of the enzyme were determined to prepare synthetic oligonucleotides as primers for the PCR. An approximate 1,000-base genomic DNA fragment thus amplified was used as the probe to clone both genomic DNA and cDNA for the enzyme. The lactonohydrolase genomic gene consists of six exons separated by five short introns. A novel type of RNA editing, in which lactonohydrolase mRNA included the insertion of guanosine and cytidine residues, was observed. The predicted amino acid sequence of the cloned lactonohydrolase cDNA showed significant similarity to those of the gluconolactonase from Zymomonas mobilis, and paraoxonases from human and rabbit, forming a unique superfamily consisting of C-O cleaving enzymes and P-O cleaving enzymes. Lactonohydrolase was expressed under the control of the lac promoter in Escherichia coli.

Glu-tRNAGln amidotransferase: A novel heterotrimeric enzyme required for correct decoding of glutamine codons during translation

The three genes, gatC, gatA, and gatB, which constitute the transcriptional unit of the Bacillus subtilis glutamyl-tRNAGln amidotransferase have been cloned. Expression of this transcriptional unit results in the production of a heterotrimeric protein that has been purified to homogeneity. The enzyme furnishes a means for formation of correctly charged Gln-tRNAGln through the transamidation of misacylated Glu-tRNAGln, functionally replacing the lack of glutaminyl-tRNA synthetase activity in Gram-positive eubacteria, cyanobacteria, Archaea, and organelles. Disruption of this operon is lethal. This demonstrates that transamidation is the only pathway to Gln-tRNAGln in B. subtilis and that glutamyl-tRNAGln amidotransferase is a novel and essential component of the translational apparatus.

Folding in vivo of a newly translated yeast cytosolic enzyme is mediated by the SSA class of cytosolic yeast Hsp70 proteins

The nature of chaperone action in the eukaryotic cytosol that assists newly translated cytosolic proteins to reach the native state has remained poorly defined. Actin, tubulin, and Gα transducin are assisted by the cytosolic chaperonin, CCT, but many other proteins, for example, ornithine transcarbamoylase (OTC), a cytosolic homotrimeric enzyme of yeast, do not require CCT action. Here, we observe that yeast cytosolic OTC is assisted to its native state by the SSA class of yeast cytosolic Hsp70 proteins. In vitro, refolding of OTC diluted from denaturant was assisted by crude yeast cytosol and ATP and found to be directed by SSA1/2. In vivo, when OTC was induced in a temperature-sensitive SSA-deficient strain, it exhibited reduced specific activity, and nonnative subunits were detected in the soluble fraction. These findings indicate that, in vivo, the Hsp70 system assists in folding at least some newly translated cytosolic enzymes, most likely functioning in a posttranslational manner.

cDNA cloning of Batis maritima methyl chloride transferase and purification of the enzyme

Methyl chloride transferase catalyzes the synthesis of methyl chloride from S-adenosine-l-methionine and chloride ion. This enzyme has been purified 2,700-fold to homogeneity from Batis maritima, a halophytic plant that grows abundantly in salt marshes. The purification of the enzyme was accomplished by a combination of ammonium sulfate fractionation, column chromatography on Sephadex G100 and adenosine-agarose, and TSK-250 size-exclusion HPLC. The purified enzyme exhibits a single band on SDS/PAGE with a molecular mass of approximately 22.5 kDa. The molecular mass of the purified enzyme was 22,474 Da as determined by matrix-associated laser desorption ionization mass spectrometry. The methylase can function in either a monomeric or oligomeric form. A 32-aa sequence of an internal fragment of the methylase was determined (GLVPGCGGGYDVVAMANPER FMVGLDIXENAL, where X represents unknown residue) by Edman degradation, and a full-length cDNA of the enzyme was obtained by rapid amplification of cDNA ends–PCR amplification of cDNA oligonucleotides. The cDNA gene contains an ORF of 690 bp encoding an enzyme of 230 aa residues having a predicted molecular mass of 25,761 Da. The disparity between the observed and calculated molecular mass suggests that the methylase undergoes posttranslational cleavage, possibly during purification. Sequence homologies suggest that the B. maritima methylase defines a new family of plant methyl transferases. A possible function for this novel methylase in halophytic plants is discussed.

Comparison of x-ray crystal structures of an acyl-enzyme intermediate of subtilisin Carlsberg formed in anhydrous acetonitrile and in water

The x-ray crystal structures of trans-cinnamoyl–subtilisin, an acyl-enzyme covalent intermediate of the serine protease subtilisin Carlsberg, have been determined to 2.2-Å resolution in anhydrous acetonitrile and in water. The cinnamoyl–subtilisin structures are virtually identical in the two solvents. In addition, their enzyme portions are nearly indistinguishable from previously determined structures of the free enzyme in acetonitrile and in water; thus, acylation in either aqueous or nonaqueous solvent causes no appreciable conformational changes. However, the locations of bound solvent molecules in the active site of the acyl- and free enzyme forms in acetonitrile and in water are distinct. Such differences in the active site solvation may contribute to the observed variations in enzymatic activities. On prolonged exposure to organic solvent or removal of interstitial solvent from the crystal lattice, the channels within enzyme crystals are shown to collapse, leading to a drop in the number of active sites accessible to the substrate. The mechanistic and preparative implications of our findings for enzymatic catalysis in organic solvents are discussed.

Identification of a gene encoding an acyl CoA:diacylglycerol acyltransferase, a key enzyme in triacylglycerol synthesis

Triacylglycerols are quantitatively the most important storage form of energy for eukaryotic cells. Acyl CoA:diacylglycerol acyltransferase (DGAT, EC 2.3.1.20) catalyzes the terminal and only committed step in triacylglycerol synthesis, by using diacylglycerol and fatty acyl CoA as substrates. DGAT plays a fundamental role in the metabolism of cellular diacylglycerol and is important in higher eukaryotes for physiologic processes involving triacylglycerol metabolism such as intestinal fat absorption, lipoprotein assembly, adipose tissue formation, and lactation. DGAT is an integral membrane protein that has never been purified to homogeneity, nor has its gene been cloned. We identified an expressed sequence tag clone that shared regions of similarity with acyl CoA:cholesterol acyltransferase, an enzyme that also uses fatty acyl CoA as a substrate. Expression of a mouse cDNA for this expressed sequence tag in insect cells resulted in high levels of DGAT activity in cell membranes. No other acyltransferase activity was detected when a variety of substrates, including cholesterol, were used as acyl acceptors. The gene was expressed in all tissues examined; during differentiation of NIH 3T3-L1 cells into adipocytes, its expression increased markedly in parallel with increases in DGAT activity. The identification of this cDNA encoding a DGAT will greatly facilitate studies of cellular glycerolipid metabolism and its regulation.

Crystal structure of SQD1, an enzyme involved in the biosynthesis of the plant sulfolipid headgroup donor UDP-sulfoquinovose

The SQD1 enzyme is believed to be involved in the biosynthesis of the sulfoquinovosyl headgroup of plant sulfolipids, catalyzing the transfer of SO3− to UDP-glucose. We have determined the structure of the complex of SQD1 from Arabidopsis thaliana with NAD+ and the putative substrate UDP-glucose at 1.6-Å resolution. Both bound ligands are completely buried within the binding cleft, along with an internal solvent cavity which is the likely binding site for the, as yet, unidentified sulfur-donor substrate. SQD1 is a member of the short-chain dehydrogenase/reductase (SDR) family of enzymes, and its structure shows a conservation of the SDR catalytic residues. Among several highly conserved catalytic residues, Thr-145 forms unusually short hydrogen bonds with both susceptible hydroxyls of UDP-glucose. A His side chain may also be catalytically important in the sulfonation.

Synergistic inhibition of tumor growth in a murine mammary adenocarcinoma model by combinational gene therapy using IL-12, pro-IL-18, and IL-1β converting enzyme cDNA

We report here that a cancer gene therapy protocol using a combination of IL-12, pro-IL-18, and IL-1β converting enzyme (ICE) cDNA expression vectors simultaneously delivered via gene gun can significantly augment antitumor effects, evidently by generating increased levels of bioactive IL-18 and consequently IFN-γ. First, we compared the levels of IFN-γ secreted by mouse splenocytes stimulated with tumor cells transfected with various test genes, including IL-12 alone; pro-IL-18 alone; pro-IL-18 and ICE; IL-12 and pro-IL-18; and IL-12, pro-IL-18, and ICE. Among these treatments, the combination of IL-12, pro-IL-18, and ICE cDNA resulted in the highest level of IFN-γ production from splenocytes in vitro, and similar results were obtained when these same treatments were delivered to the skin of a mouse by gene gun and IFN-γ levels were measured at the skin transfection site in vivo. Furthermore, the triple gene combinatorial gene therapy protocol was the most effective among all tested groups at suppressing the growth of TS/A (murine mammary adenocarcinoma) tumors previously implanted intradermally at the skin site receiving DNA transfer by gene gun on days 6, 8, 10, and 12 after tumor implantation. Fifty percent of mice treated with the combined three-gene protocol underwent complete tumor regression. In vivo depletion experiments showed that this antitumor effect was CD8+ T cell-mediated and partially IFN-γ-dependent. These results suggest that a combinatorial gene therapy protocol using a mixture of IL-12, pro-IL-18, and ICE cDNAs can confer potent antitumor activities against established TS/A tumors via cytotoxic CD8+ T cells and IFN-γ-dependent pathways.

Serine racemase: A glial enzyme synthesizing d-serine to regulate glutamate-N-methyl-d-aspartate neurotransmission

Although d amino acids are prominent in bacteria, they generally are thought not to occur in mammals. Recently, high levels of d-serine have been found in mammalian brain where it activates glutamate/N-methyl-d-aspartate receptors by interacting with the “glycine site” of the receptor. Because amino acid racemases are thought to be restricted to bacteria and insects, the origin of d-serine in mammals has been puzzling. We now report cloning and expression of serine racemase, an enzyme catalyzing the formation of d-serine from l-serine. Serine racemase is a protein representing an additional family of pyridoxal-5′ phosphate-dependent enzymes in eukaryotes. The enzyme is enriched in rat brain where it occurs in glial cells that possess high levels of d-serine in vivo. Occurrence of serine racemase in the brain demonstrates the conservation of d-amino acid metabolism in mammals with implications for the regulation of N-methyl-d-aspartate neurotransmission through glia-neuronal interactions.