Determinants of Endogenous Ligand Specificity Divergence among Metabotropic Glutamate Receptors

To determine the structural origins of diverse ligand response specificities among metabotropic glutamate receptors (mGluRs), we combined computational approaches with mutagenesis and ligand response assays to identify specificity-determining residues in the group I receptor, mGluR1, and the group III receptors, mGluR4 and mGluR7. Among these, mGluR1 responds to l-glutamate effectively, whereas it binds weakly to another endogenous ligand, l-serine-O-phosphate (l-SOP), which antagonizes the effects of l-glutamate. In contrast, mGluR4 has in common with other group III mGluR that it is activated with higher potency and efficacy by l-SOP. mGluR7 differs from mGluR4 and other group III mGluR in that l-glutamate and l-SOP activate it with low potency and efficacy. Enhanced versions of the evolutionary trace (ET) algorithm were used to identify residues that when swapped between mGluR1 and mGluR4 increased the potency of l-SOP inhibition relative to the potency of l-glutamate activation in mGluR1 mutants and others that diminished the potency/efficacy of l-SOP for mGluR4 mutants. In addition, combining ET identified swaps from mGluR4 with one identified by computational docking produced mGluR7 mutants that respond with dramatically enhanced potency/efficacy to l-SOP. These results reveal that an early functional divergence between group I/II and group III involved variation at positions primarily at allosteric sites located outside of binding pockets, whereas a later divergence within group III occurred through sequence variation both at the ligand-binding pocket and at loops near the dimerization interface and interlobe hinge region. They also demonstrate the power of ET for identifying allosteric determinants of evolutionary importance.     

Selectivity and Evolutionary Divergence of Metabotropic Glutamate Receptors for Endogenous Ligands and G Proteins Coupled to Phospholipase C or TRP Channels

To define the upstream and downstream signaling specificities of metabotropic glutamate receptors (mGluR), we have examined the ability of representative mGluR of group I, II, and III to be activated by endogenous amino acids and catalyze activation of G proteins coupled to phospholipase C (PLC), or activation of G_i/o proteins coupled to the ion channel TRPC4β. Fluorescence-based assays have allowed us to observe interactions not previously reported or clearly identified. We have found that the specificity for endogenous amino acids is remarkably stringent. Even at millimolar levels, structurally similar compounds do not elicit significant activation. As reported previously, the clear exception is l-serine-O-phosphate (l-SOP), which strongly activates group III mGluR, especially mGluR4,-6,-8 but not group I or II mGluR. Whereas l-SOP cannot activate mGluR1 or mGluR2, it acts as a weak antagonist for mGluR1 and a potent antagonist for mGluR2, suggesting that co-recognition of l-glutamate and l-SOP arose early in evolution, and was followed later by divergence of group I and group II mGluR versus group III in l-SOP responses. mGluR7 has low affinity and efficacy for activation by both l-glutamate and l-SOP. Molecular docking studies suggested that residue 74 corresponding to lysine in mGluR4 and asparagine in mGluR7 might play a key role, and, indeed, mutagenesis experiments demonstrated that mutating this residue to lysine in mGluR7 enhances the potency of l-SOP. Experiments with pertussis toxin and dominant-negative Gα_i/o proteins revealed that mGluR1 couples strongly to TRPC4β through Gα_i/o, in addition to coupling to PLC through Gα_q/11.     

FBXO22 Protein Is Required for Optimal Synthesis of the N-Methyl-d-Aspartate (NMDA) Receptor Coagonist d-Serine

d-Serine is a physiological activator of NMDA receptors (NMDARs) in the nervous system that mediates several NMDAR-mediated processes ranging from normal neurotransmission to neurodegeneration. d-Serine is synthesized from l-serine by serine racemase (SR), a brain-enriched enzyme. However, little is known about the regulation of d-serine synthesis. We now demonstrate that the F-box only protein 22 (FBXO22) interacts with SR and is required for optimal d-serine synthesis in cells. Although FBXO22 is classically associated with the ubiquitin system and is recruited to the Skip1-Cul1-F-box E3 complex, SR interacts preferentially with free FBXO22 species. In vivo ubiquitination and SR half-life determination indicate that FBXO22 does not target SR to the proteasome system. FBXO22 primarily affects SR subcellular localization and seems to increase d-serine synthesis by preventing the association of SR to intracellular membranes. Our data highlight an atypical role of FBXO22 in enhancing d-serine synthesis that is unrelated to its classical effects as a component of the ubiquitin-proteasome degradation pathway.     

Serine Racemase Regulated by Binding to Stargazin and PSD-95: POTENTIAL N-METHYL-d-ASPARTATE-α-AMINO-3-HYDROXY-5-METHYL-4-ISOXAZOLEPROPIONIC ACID (NMDA-AMPA) GLUTAMATE NEUROTRANSMISSION CROSS-TALK*

d-Serine, an endogenous co-agonist for the glycine site of the synaptic NMDA glutamate receptor, regulates synaptic plasticity and is implicated in schizophrenia. Serine racemase (SR) is the enzyme that converts l-serine to d-serine. In this study, we demonstrate that SR interacts with the synaptic proteins, postsynaptic density protein 95 (PSD-95) and stargazin, forming a ternary complex. SR binds to the PDZ3 domain of PSD-95 through the PDZ domain ligand at its C terminus. SR also binds to the C terminus of stargazin, which facilitates the cell membrane localization of SR and inhibits its activity. AMPA receptor activation internalizes SR and disrupts its interaction with stargazin, therefore derepressing SR activity, leading to more d-serine production and potentially facilitating NMDA receptor activation. These interactions regulate the enzymatic activity as well as the intracellular localization of SR, potentially coupling the activities of NMDA and AMPA receptors. This shuttling of a neurotransmitter synthesizing enzyme between two receptors appears to be a novel mode of synaptic regulation.     

A Novel Type of Peptidoglycan-binding Domain Highly Specific for Amidated d-Asp Cross-bridge,Identified in Lactobacillus casei Bacteriophage Endolysins

Peptidoglycan hydrolases (PGHs) are responsible for bacterial cell lysis. Most PGHs have a modular structure comprising a catalytic domain and a cell wall-binding domain (CWBD). PGHs of bacteriophage origin, called endolysins, are involved in bacterial lysis at the end of the infection cycle. We have characterized two endolysins, Lc-Lys and Lc-Lys-2, identified in prophages present in the genome of Lactobacillus casei BL23. These two enzymes have different catalytic domains but similar putative C-terminal CWBDs. By analyzing purified peptidoglycan (PG) degradation products, we showed that Lc-Lys is an N-acetylmuramoyl-l-alanine amidase, whereas Lc-Lys-2 is a γ-d-glutamyl-l-lysyl endopeptidase. Remarkably, both lysins were able to lyse only Gram-positive bacterial strains that possess PG with d-Ala⁴→d-Asx-l-Lys³ in their cross-bridge, such as Lactococcus casei, Lactococcus lactis, and Enterococcus faecium. By testing a panel of L. lactis cell wall mutants, we observed that Lc-Lys and Lc-Lys-2 were not able to lyse mutants with a modified PG cross-bridge, constituting d-Ala⁴→l-Ala-(l-Ala/l-Ser)-l-Lys³; moreover, they do not lyse the L. lactis mutant containing only the nonamidated d-Asp cross-bridge, i.e. d-Ala⁴→d-Asp-l-Lys³. In contrast, Lc-Lys could lyse the ampicillin-resistant E. faecium mutant with 3→3 l-Lys³-d-Asn-l-Lys³ bridges replacing the wild-type 4→3 d-Ala⁴-d-Asn-l-Lys³ bridges. We showed that the C-terminal CWBD of Lc-Lys binds PG containing mainly d-Asn but not PG with only the nonamidated d-Asp-containing cross-bridge, indicating that the CWBD confers to Lc-Lys its narrow specificity. In conclusion, the CWBD characterized in this study is a novel type of PG-binding domain targeting specifically the d-Asn interpeptide bridge of PG.     

Third Transmembrane Domain of the Adrenocorticotropic Receptor Is Critical for Ligand Selectivity and Potency

The ACTH receptor, known as the melanocortin-2 receptor (MC2R), plays an important role in regulating and maintaining adrenocortical function. MC2R is a subtype of the melanocortin receptor (MCR) family and has unique characteristics among MCRs. Endogenous ACTH is the only endogenous agonist for MC2R, whereas the melanocortin peptides α-, β-, and γ-melanocyte-stimulating hormone and ACTH are full agonists for all other MCRs. In this study, we examined the molecular basis of MC2R responsible for ligand selectivity using ACTH analogs and MC2R mutagenesis. Our results indicate that substitution of Phe⁷ with d-Phe or d-naphthylalanine (d-Nal(2′)) in ACTH(1–24) caused a significant decrease in ligand binding affinity and potency. Substitution of Phe⁷ with d-Nal(2′) in ACTH(1–24) did not switch the ligand from agonist to antagonist at MC2R, which was observed in MC3R and MC4R. Substitution of Phe⁷ with d-Phe⁷ in ACTH(1–17) resulted in the loss of ligand binding and activity. Molecular analysis of MC2R indicated that only mutation of the third transmembrane domain of MC2R resulted in a decrease in d-Phe ACTH binding affinity and potency. Our results suggest that Phe⁷ in ACTH plays an important role in ligand selectivity and that the third transmembrane domain of MC2R is crucial for ACTH selectivity and potency.     

Non-repair Pathways for Minimizing Protein Isoaspartyl Damage in the Yeast Saccharomyces cerevisiae

The spontaneous degradation of asparaginyl and aspartyl residues to isoaspartyl residues is a common type of protein damage in aging organisms. Although the protein-l-isoaspartyl (d-aspartyl) O-methyltransferase (EC 2.1.1.77) can initiate the repair of l-isoaspartyl residues to l-aspartyl residues in most organisms, no gene homolog or enzymatic activity is present in the budding yeast Saccharomyces cerevisiae. Therefore, we used biochemical approaches to elucidate how proteins containing isoaspartyl residues are metabolized in this organism. Surprisingly, the level of isoaspartyl residues in yeast proteins (50–300 pmol of isoaspartyl residues/mg of protein extract) is comparable with organisms with protein-l-isoaspartyl (d-aspartyl) O-methyltransferase, suggesting a novel regulatory pathway. Interfering with common protein quality control mechanisms by mutating and inhibiting the proteasomal and autophagic pathways in vivo did not increase isoaspartyl residue levels compared with wild type or uninhibited cells. However, the inhibition of metalloproteases in in vitro aging experiments by EDTA resulted in an ∼3-fold increase in the level of isoaspartyl-containing peptides. Characterization by mass spectrometry of these peptides identified several proteins involved in metabolism as targets of isoaspartyl damage. Further analysis of these peptides revealed that many have an N-terminal isoaspartyl site and originate from proteins with short half-lives. These results suggest that one or more metalloproteases participate in limiting isoaspartyl formation by robust proteolysis.     

Recharacterization of the mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase as 4-oxo-l-proline reductase (EC 1.1.1.104)

Early studies revealed that chicken embryos incubated with a rare analog of l-proline, 4-oxo-l-proline, showed increased levels of the metabolite 4-hydroxy-l-proline. In 1962, 4-oxo-l-proline reductase, an enzyme responsible for the reduction of 4-oxo-l-proline, was partially purified from rabbit kidneys and characterized biochemically. However, only recently was the molecular identity of this enzyme solved. Here, we report the purification from rat kidneys, identification, and biochemical characterization of 4-oxo-l-proline reductase. Following mass spectrometry analysis of the purified protein preparation, the previously annotated mammalian cytosolic type 2 (R)-β-hydroxybutyrate dehydrogenase (BDH2) emerged as the only candidate for the reductase. We subsequently expressed rat and human BDH2 in Escherichia coli, then purified it, and showed that it catalyzed the reversible reduction of 4-oxo-l-proline to cis-4-hydroxy-l-proline via chromatographic and tandem mass spectrometry analysis. Specificity studies with an array of compounds carried out on both enzymes showed that 4-oxo-l-proline was the best substrate, and the human enzyme acted with 12,500-fold higher catalytic efficiency on 4-oxo-l-proline than on (R)-β-hydroxybutyrate. In addition, human embryonic kidney 293T (HEK293T) cells efficiently metabolized 4-oxo-l-proline to cis-4-hydroxy-l-proline, whereas HEK293T BDH2 KO cells were incapable of producing cis-4-hydroxy-l-proline. Both WT and KO HEK293T cells also produced trans-4-hydroxy-l-proline in the presence of 4-oxo-l-proline, suggesting that the latter compound might interfere with the trans-4-hydroxy-l-proline breakdown in human cells. We conclude that BDH2 is a mammalian 4-oxo-l-proline reductase that converts 4-oxo-l-proline to cis-4-hydroxy-l-proline and not to trans-4-hydroxy-l-proline, as originally thought. We also hypothesize that this enzyme may be a potential source of cis-4-hydroxy-l-proline in mammalian tissues.     

The biological sulphation of l-tyrosyl peptides

1. A rat-liver supernatant preparation can achieve the biological O-sulphation of l-tyrosylglycine and l-tyrosyl-l-alanine at pH7·0. 2. The optimum concentrations of l-tyrosylglycine and l-tyrosyl-l-alanine in this system are 50mm and 60mm respectively. 3. l-Tyrosylglycine yields two sulphated products, whereas l-tyrosyl-l-alanine yields three sulphated products, when used as acceptor for sulphate in the rat-liver system. 4. With both substrates, one of the sulphated products has been identified as the O-sulphate ester of the corresponding parent peptide.     

Biosynthesis of l-tyrosine O-sulphate from the methyl and ethyl esters of l-tyrosine

1. Rat-liver supernatant preparations are capable of achieving the biological sulphation of l-tyrosine methyl ester, the reaction proceeding maximally at a substrate concentration of 30 mm and at pH 7·0. 2. Two sulphated products are formed, one of which has been identified as l-tyrosine O-sulphate. On the basis of indirect evidence the other product can be assumed to be l-tyrosine O-sulphate methyl ester. 3. An enzyme present in rat-liver supernatant preparations is capable of converting l-tyrosine O-sulphate methyl ester into l-tyrosine O-sulphate. This enzyme is inhibited by l-tyrosine methyl ester. 4. l-Tyrosine ethyl ester also yields two sulphated products when used as an acceptor in the liver sulphating system. One of these has been identified chromatographically as l-tyrosine O-sulphate and the other may be presumed to be l-tyrosine O-sulphate ethyl ester.     

Amino Acid and vitamin requirements of several bacteroides strains

Nutritional studies were performed on nine Bacteroides strains, by use of the methodology and media of anaerobic rumen microbiology. Ristella perfoetens CCI required l-arginine hydrochloride, l-tryptophan, l-leucine, l-histidine hydrochloride, l-cysteine hydrochloride, dl-valine, dl-tyrosine, and the vitamin calcium-d-pantothenate, since scant turbidity developed in media without these nutrients. R. perfoetens was stimulated by glycine, dl-lysine hydrochloride, dl-isoleucine, l-proline, l-glutamic acid, dl-alanine, dl-phenylalanine, dl-methionine, and the vitamins nicotinamide and p-aminobenzoic acid, since maximal turbidity developed more slowly in media without these nutrients than in complete medium. Medium A-23, which was devised for R. perfoetens, contained salts, 0.0002% nicotinamide and calcium d-pantothenate, 0.00001% p-aminobenzoic acid, 0.044% l-tryptophan, 0.09% l-glutamic acid, and 0.1% of the other 13 amino acids listed above. Zuberella clostridiformis and seven strains of R. pseudoinsolita did not require vitamins, and showed no absolute requirement for any one amino acid. Various strains produced maximal turbidity more slowly in media deficient in l-proline, glycine, l-glutamic acid, dl-serine, l-histidine hydrochloride, dl-alanine, or l-cysteine hydrochloride, than in complete medium. These eight strains grew optimally in medium A-23 plus 0.1% dl-serine but without vitamins.     

Identification of an l-Arabinose Reductase Gene in Aspergillus niger and Its Role in l-Arabinose Catabolism

The first enzyme in the pathway for l-arabinose catabolism in eukaryotic microorganisms is a reductase, reducing l-arabinose to l-arabitol. The enzymes catalyzing this reduction are in general nonspecific and would also reduce d-xylose to xylitol, the first step in eukaryotic d-xylose catabolism. It is not clear whether microorganisms use different enzymes depending on the carbon source. Here we show that Aspergillus niger makes use of two different enzymes. We identified, cloned, and characterized an l-arabinose reductase, larA, that is different from the d-xylose reductase, xyrA. The larA is up-regulated on l-arabinose, while the xyrA is up-regulated on d-xylose. There is however an initial up-regulation of larA also on d-xylose but that fades away after about 4 h. The deletion of the larA gene in A. niger results in a slow growth phenotype on l-arabinose, whereas the growth on d-xylose is unaffected. The l-arabinose reductase can convert l-arabinose and d-xylose to their corresponding sugar alcohols but has a higher affinity for l-arabinose. The K_m for l-arabinose is 54 ± 6 mm and for d-xylose 155 ± 15 mm.     

An Immunomodulating Motif of the HIV-1 Fusion Protein Is Chirality-independent: IMPLICATIONS FOR ITS MODE OF ACTION*

An immunosuppressive motif was recently found within the HIV-1 gp41 fusion protein (termed immunosuppressive loop-associated determinant core motif (ISLAD CM)). Peptides containing the motif interact with the T-cell receptor (TCR) complex; however, the mechanism by which the motif exerts its immunosuppressive activity is yet to be determined. Recent studies showed that interactions between protein domains in the membrane milieu are not always sterically controlled. Therefore, we utilized the unique membrane leniency toward association between d- and l-stereoisomers to investigate the detailed mechanism by which ISLAD CM inhibits T-cell activation. We show that a d-enantiomer of ISLAD CM (termed ISLAD d-CM) inhibited the proliferation of murine myelin oligodendrocyte glycoprotein (MOG)-(35–55)-specific line T-cells to the same extent as the l-motif form. Moreover, the d- and l-forms preferentially bound spleen-derived T-cells over B-cells by 13-fold. Furthermore, both forms of ISLAD CM co-localized with the TCR on activated T-cells and interacted with the transmembrane domain of the TCR. FRET experiments revealed the importance of basic residues for the interaction between ISLAD CM forms and the TCR transmembrane domain. Ex vivo studies demonstrated that ISLAD d-CM administration inhibited the proliferation (72%) and proinflammatory cytokine secretion of pathogenic MOG(35–55)-specific T-cells. This study provides insights into the immunosuppressive mechanism of gp41 and demonstrates that chirality-independent interactions in the membrane can take place in diverse biological systems. Apart from HIV pathogenesis, the d-peptide reported herein may serve as a potential tool for treating T-cell-mediated pathologies.     

The Cell Shape-determining Csd6 Protein from Helicobacter pylori Constitutes a New Family of l,d-Carboxypeptidase

Helicobacter pylori causes gastrointestinal diseases, including gastric cancer. Its high motility in the viscous gastric mucosa facilitates colonization of the human stomach and depends on the helical cell shape and the flagella. In H. pylori, Csd6 is one of the cell shape-determining proteins that play key roles in alteration of cross-linking or by trimming of peptidoglycan muropeptides. Csd6 is also involved in deglycosylation of the flagellar protein FlaA. To better understand its function, biochemical, biophysical, and structural characterizations were carried out. We show that Csd6 has a three-domain architecture and exists as a dimer in solution. The N-terminal domain plays a key role in dimerization. The middle catalytic domain resembles those of l,d-transpeptidases, but its pocket-shaped active site is uniquely defined by the four loops I to IV, among which loops I and III show the most distinct variations from the known l,d-transpeptidases. Mass analyses confirm that Csd6 functions only as an l,d-carboxypeptidase and not as an l,d-transpeptidase. The d-Ala-complexed structure suggests possible binding modes of both the substrate and product to the catalytic domain. The C-terminal nuclear transport factor 2-like domain possesses a deep pocket for possible binding of pseudaminic acid, and in silico docking supports its role in deglycosylation of flagellin. On the basis of these findings, it is proposed that H. pylori Csd6 and its homologs constitute a new family of l,d-carboxypeptidase. This work provides insights into the function of Csd6 in regulating the helical cell shape and motility of H. pylori.     

Role of Conformational Dynamics in α-Amino-3-hydroxy-5-methylisoxazole-4-propionic Acid (AMPA) Receptor Partial Agonism

We have investigated the range of cleft closure conformational states that the agonist-binding domains of the α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptors occupy when bound to a series of willardiine derivatives using single-molecule FRET. These studies show that the agonist-binding domain exhibits varying degrees of dynamics when bound to the different willardiines with differing efficacies. The chlorowillardiine- and nitrowillardiine-bound form of the agonist-binding domain probes a narrower range of cleft closure states relative to the iodowillardiine bound form of the protein, with the antagonist (αS)-α-amino-3-[(4-carboxyphenyl)methyl]-3,4-dihydro-2,4-dioxo-1(2H)-pyrimidinepropanoic acid (UBP-282)-bound form exhibiting the widest range of cleft closure states. Additionally, the average cleft closure follows the order UBP-282 > iodowillardiine > chlorowillardiine > nitrowillardiine-bound forms of agonist-binding domain. These single-molecule FRET data, along with our previously reported data for the glutamate-bound forms of wild type and T686S mutant proteins, show that the mean currents under nondesensitizing conditions can be directly correlated to the fraction of the agonist-binding domains in the “closed” cleft conformation. These results indicate that channel opening in the AMPA receptors is controlled by both the ability of the agonist to induce cleft closure and the dynamics of the agonist-binding domain when bound to the agonist.     

Substrate Specificity of a Mannose-6-Phosphate Isomerase from Bacillus subtilis and Its Application in the Production of l-Ribose

The uncharacterized gene previously proposed as a mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme was observed at pH 7.5 and 40°C in the presence of 0.5 mM Co²⁺. The isomerization activity was specific for aldose substrates possessing hydroxyl groups oriented in the same direction at the C-2 and C-3 positions, such as the d and l forms of ribose, lyxose, talose, mannose, and allose. The enzyme exhibited the highest activity for l-ribulose among all pentoses and hexoses. Thus, l-ribose, as a potential starting material for many l-nucleoside-based pharmaceutical compounds, was produced at 213 g/liter from 300-g/liter l-ribulose by mannose-6-phosphate isomerase at 40°C for 3 h, with a conversion yield of 71% and a volumetric productivity of 71 g liter⁻¹ h⁻¹.l-Ribose is a potential starting material for the synthesis of many l-nucleoside-based pharmaceutical compounds, and it is not abundant in nature (5, 19). l-Ribose has been produced mainly by chemical synthesis from l-arabinose, l-xylose, d-glucose, d-galactose, d-ribose, or d-mannono-1,4-lactone (2, 17, 23). Biological l-ribose manufacture has been investigated using ribitol or l-ribulose. Recently, l-ribose was produced from ribitol by a recombinant Escherichia coli containing an NAD-dependent mannitol-1-dehydrogenase (MDH) with a 55% conversion yield when 100 g/liter ribitol was used in a 72-h fermentation (18). However, the volumetric productivity of l-ribose in the fermentation is 28-fold lower than that of the chemical method synthesized from l-arabinose (8). l-Ribulose has been biochemically converted from l-ribose using an l-ribose isomerase from an Acinetobacter sp. (9), an l-arabinose isomerase mutant from Escherichia coli (4), a d-xylose isomerase mutant from Actinoplanes missouriensis (14), and a d-lyxose isomerase from Cohnella laeviribosi (3), indicating that l-ribose can be produced from l-ribulose by these enzymes. However, the enzymatic production of l-ribulose is slow, and the enzymatic production of l-ribose from l-ribulose has been not reported.Sugar phosphate isomerases, such as ribose-5-phosphate isomerase, glucose-6-phosphate isomerase, and galactose-6-phosphate isomerase, work as general aldose-ketose isomerases and are useful tools for producing rare sugars, because they convert the substrate sugar phosphates and the substrate sugars without phosphate to have a similar configuration (11, 12, 21, 22). l-Ribose isomerase from an Acinetobacter sp. (9) and d-lyxose isomerase from C. laeviribosi (3) had activity with l-ribose, d-lyxose, and d-mannose. Thus, we can apply mannose-6-phosphate (EC 5.3.1.8) isomerase to the production of l-ribose, because there are no sugar phosphate isomerases relating to l-ribose and d-lyxose. The production of the expensive sugar l-ribose (bulk price, $1,000/kg) from the rare sugar l-ribulose by mannose-6-phosphate isomerase may prove to be a valuable industrial process, because we have produced l-ribulose from the cheap sugar l-arabinose (bulk price, $50/kg) using the l-arabinose isomerase from Geobacillus thermodenitrificans (20) (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Schematic representation for the production of l-ribulose from l-arabinose by G. thermodenitrificans l-arabinose isomerase and the production of l-ribose from l-ribulose by B. subtilis mannose-6-phosphate isomerase.In this study, the gene encoding mannose-6-phosphate isomerase from Bacillus subtilis was cloned and expressed in E. coli. The substrate specificity of the recombinant enzyme for various aldoses and ketoses was investigated, and l-ribulose exhibited the highest activity among all pentoses and hexoses. Therefore, mannose-6-phosphate isomerase was applied to the production of l-ribose from l-ribulose.     

Mechanism of Allosteric Inhibition of N-Acetyl-L-glutamate Synthase by L-Arginine

N-Acetylglutamate synthase (NAGS) catalyzes the first committed step in l-arginine biosynthesis in plants and micro-organisms and is subject to feedback inhibition by l-arginine. This study compares the crystal structures of NAGS from Neisseria gonorrhoeae (ngNAGS) in the inactive T-state with l-arginine bound and in the active R-state complexed with CoA and l-glutamate. Under all of the conditions examined, the enzyme consists of two stacked trimers. Each monomer has two domains: an amino acid kinase (AAK) domain with an AAK-like fold but lacking kinase activity and an N-acetyltransferase (NAT) domain homologous to other GCN5-related transferases. Binding of l-arginine to the AAK domain induces a global conformational change that increases the diameter of the hexamer by ∼10 Å and decreases its height by ∼20Å_. AAK dimers move 5Å outward along their 2-fold axes, and their tilt relative to the plane of the hexamer decreases by ∼4°. The NAT domains rotate ∼109° relative to AAK domains enabling new interdomain interactions. Interactions between AAK and NAT domains on different subunits also change. Local motions of several loops at the l-arginine-binding site enable the protein to close around the bound ligand, whereas several loops at the NAT active site become disordered, markedly reducing enzymatic specific activity.l-Arginine biosynthesis in most micro-organisms and plants involves the initial acetylation of l-glutamate by N-acetylglutamate synthase (NAGS, EC 2.3.1.1)² to produce N-acetylglutamate (NAG). NAG is then converted by NAG kinase (NAGK, EC 2.7.2.8) to NAG-phosphate and subsequently to N-acetylornithine (1, 2). Two alternative reactions are used to remove the acetyl group from acetylornithine. The linear pathway uses N-acetylornithine deacetylase (EC 3.5.1.16) to catalyze the metal-dependent hydrolysis of the acetyl group to form l-ornithine and acetate, whereas the acetyl recycling pathway transfers the acetyl group from N-acetylornithine to l-glutamate, producing l-ornithine and NAG. This reaction is catalyzed by ornithine acetyltransferase (EC 2.3.1.35).In the linear pathway, NAGS is the only target of feedback inhibition by l-arginine. In contrast, in the acetyl cycling pathway l-arginine may inhibit NAGS and NAGK or ornithine acetyltransferase (3). Structure determinations of l-arginine-insensitive (4) and l-arginine-sensitive NAGKs (5) provided insights into the structural basis of l-arginine inhibition of NAGK. l-Arginine-insensitive Escherichia coli (ec) NAGK is a homodimer (4), whereas l-arginine-sensitive NAGKs from Thermotoga maritima (tm) and Pseudomonas aeruginosa (pa) are hexamers formed by pair-wise interlacing of the N-terminal helices of three ecNAGK-like dimers, to create a second type of dimer interface. l-Arginine binding to a site close to the C terminus induces global conformational changes that expands the ring by ∼8 Å and decreases the tilt of the ecNAGK-like dimers relative to the plane of the ring by ∼6°. The inhibition mechanism was proposed to involve the enlargement of an active site located close to the l-arginine-binding site.Because of the sequence similarity between NAGK and NAGS, it was speculated that they may have similar l-arginine-binding sites and hexameric ring structures (5). However, our recent structural determination of NAGS from Neisseria gonorrhoeae (ng) revealed the active site to be located in the NAT domain, >25 Å away from the proposed l-arginine-binding site (6). Therefore, the allosteric mechanism of NAGS is likely to be different from that of l-arginine-sensitive NAGKs. Here we compare the structures of ngNAGS in the inactive T-state with l-arginine bound and in the R-state complexed with CoA and l-glutamate and determine the structural basis for the allosteric inhibition of NAGS by l-arginine.     

Probing the Molecular Determinant for the Catalytic Efficiency of l-Arabinose Isomerase from Bacillus licheniformis

Bacillus licheniformis l-arabinose isomerase (l-AI) is distinguished from other l-AIs by its high degree of substrate specificity for l-arabinose and its high turnover rate. A systematic strategy that included a sequence alignment-based first screening of residues and a homology model-based second screening, followed by site-directed mutagenesis to alter individual screened residues, was used to study the molecular determinants for the catalytic efficiency of B. licheniformis l-AI. One conserved amino acid, Y333, in the substrate binding pocket of the wild-type B. licheniformis l-AI was identified as an important residue affecting the catalytic efficiency of B. licheniformis l-AI. Further insights into the function of residue Y333 were obtained by replacing it with other aromatic, nonpolar hydrophobic amino acids or polar amino acids. Replacing Y333 with the aromatic amino acid Phe did not alter catalytic efficiency toward l-arabinose. In contrast, the activities of mutants containing a hydrophobic amino acid (Ala, Val, or Leu) at position 333 decreased as the size of the hydrophobic side chain of the amino acid decreased. However, mutants containing hydrophilic and charged amino acids, such as Asp, Glu, and Lys, showed almost no activity with l-arabinose. These data and a molecular dynamics simulation suggest that Y333 is involved in the catalytic efficiency of B. licheniformis l-AI.l-Arabinose isomerase (l-AI) is an enzyme that mediates in vivo isomerization between l-arabinose and l-ribulose as well as in vitro isomerization of d-galactose and d-tagatose (20). l-Ribulose (l-erythro-pentulose) is a rare and expensive ketopentose sugar (1) that can be used as a precursor for the production of other rare sugars of high market value, such as l-ribose. Despite being a common metabolic intermediate in different organisms, l-ribulose is scarce in nature. The market for rare and unnatural sugars has been growing, especially in the sweetener and pharmaceutical industries. For example, several modified nucleosides derived from l-sugars have been shown to act as potent antiviral agents and are also useful in antigen therapy. Derivatives of rare sugars have also been used as agents against hepatitis B virus and human immunodeficiency virus (2, 22).For these reasons, interest in the enzymology of rare sugars has also been increasing. Various forms of l-AI from a variety of organisms have been characterized, and some have shown potential for industrial use. Several highly thermotolerant enzyme forms from Thermotoga maritima (12), Thermotoga neapolitana (10), Bacillus stearothermophilus (18), Thermoanaerobacter mathranii (9), and Lactobacillus plantarum (5) have been characterized previously. All of these reported l-AIs tend to have broad specificity, although a few l-AIs with high degrees of substrate specificity for l-arabinose have also been documented.The enzyme properties of l-AIs have been examined by engineering several forms by error-prone PCR and site-directed mutagenesis. Galactose conversion was reportedly enhanced 20% following site-directed introduction of a double mutation (C450S-N475K) into l-AI (16). Error-prone PCR manipulation of l-AI from Geobacillus stearothermophilus resulted in a shift in temperature specificity from 60 to 65°C and increased isomerization activity (11). All of these previously reported mutational studies have been aimed at improving enzymatic properties for industrial application. However, even though the three-dimensional (3D) structure of Escherichia coli l-AI has been determined previously (15), few new structural studies have been performed to decipher the reaction mechanism of this enzyme. Rhimi et al. (19) have reported an important role for D308, F329, E351, and H446 in catalysis, as indicated by findings from site-directed mutagenesis. Nonetheless, detailed analysis of the important molecular determinants controlling the catalytic activities of the l-AIs is still lacking.Previously, we have reported the cloning and characterization of a novel l-AI from Bacillus licheniformis (17). This enzyme can be distinguished from other l-AIs by its wide pH range, high degree of substrate specificity for l-arabinose, and extremely high turnover rate. In the present paper, we report the identification of an important amino acid residue responsible for the catalytic efficiency of l-AIs, as determined by a systematic screening process composed of sequence alignment and molecular dynamics (MD) simulation, followed by site-directed mutagenesis. Using the crystal structure of E. coli l-AI as a template, we have built a 3D model of B. licheniformis l-AI. Analysis of the 3D model of B. licheniformis l-AI docked with l-arabinose, followed by a systematic screening process, showed that Y333 interacted with the substrate, suggesting that this residue in B. licheniformis l-AI may be essential for catalysis. We further characterized the role of Y333 in B. licheniformis l-AI binding of and catalytic efficiency for l-arabinose.     

Deciphering a Molecular Mechanism of Neonatal Diabetes Mellitus by the Chemical Synthesis of a Protein Diastereomer, [d-AlaB8]Human Proinsulin

Misfolding of proinsulin variants in the pancreatic β-cell, a monogenic cause of permanent neonatal-onset diabetes mellitus, provides a model for a disease of protein toxicity. A hot spot for such clinical mutations is found at position B8, conserved as glycine within the vertebrate insulin superfamily. We set out to investigate the molecular basis of the aberrant properties of a proinsulin clinical mutant in which residue Gly^B8 is replaced by Ser^B8. Modular total chemical synthesis was used to prepare the wild-type [Gly^B8]proinsulin molecule and three analogs: [d-Ala^B8]proinsulin, [l-Ala^B8]proinsulin, and the clinical mutant [l-Ser^B8]proinsulin. The protein diastereomer [d-Ala^B8]proinsulin produced higher folding yields at all pH values compared with the wild-type proinsulin and the other two analogs, but showed only very weak binding to the insulin receptor. The clinical mutant [l-Ser^B8]proinsulin impaired folding at pH 7.5 even in the presence of protein-disulfide isomerase. Surprisingly, although [l-Ser^B8]proinsulin did not fold well under the physiological conditions investigated, once folded the [l-Ser^B8]proinsulin protein molecule bound to the insulin receptor more effectively than wild-type proinsulin. Such paradoxical gain of function (not pertinent in vivo due to impaired secretion of the mutant insulin) presumably reflects induced fit in the native mechanism of hormone-receptor engagement. This work provides insight into the molecular mechanism of a clinical mutation in the insulin gene associated with diabetes mellitus. These results dramatically illustrate the power of total protein synthesis, as enabled by modern chemical ligation methods, for the investigation of protein folding and misfolding.