Single mutation alters the substrate specificity of L-amino acid ligase.

L-Amino acid ligase (Lal) catalyzes the formation of a dipeptide from two L-amino acids in an ATP-dependent manner and belongs to the ATP-grasp superfamily. Bacillus subtilis YwfE, the first identified Lal, produces the dipeptide antibiotic bacilysin, which consists of L-Ala and L-anticapsin. Its substrate specificity is restricted to smaller amino acids such as L-Ala for the N-terminal end of the dipeptide, whereas a wide range of hydrophobic amino acids including L-Phe and L-Met are recognized for the C-terminal end in vitro. We determined the crystal structures of YwfE with bound ADP-Mg(2+)-Pi and ADP-Mg(2+)-L-Ala at 1.9 and 2.0 Å resolutions, respectively. On the basis of these structures, we generated point mutants of residues that are considered to participate in the recognition of L-Ala and measured their ATPase activity. The conserved Arg328 is suggested to be a crucial residue for L-Ala recognition and catalysis. The mutation of Trp332 to Ala caused the enzyme to hydrolyze ATP, even in the absence of l-Ala, and the structure of this mutant protein appeared to show a cavity in the N-terminal substrate-binding pocket. These results suggest that Trp332 plays a key role in restricting the substrate specificity to smaller amino acids such as L-Ala. Moreover, Trp332 mutants can alter the substrate specificity and activity depending on the size and shape of substituted amino acids. These observations provide sufficient scope for the rational design of Lal to produce desirable dipeptides. We propose that the positioning of the conserved Arg residue in Lal is important for enantioselective recognition of L-amino acids.

Role of positions e and g in the fibrous assembly formation of an amphipathic α-helix-forming polypeptide.

We previously characterized α3, a polypeptide that has a three times repeated sequence of seven amino acids (abcdefg: LETLAKA) and forms fibrous assemblies composed of amphipathic α-helices. Upon comparison of the amino acid sequences of α3 with other α-helix forming polypeptides, we proposed that the fibrous assemblies were formed due to the alanine (Ala) residues at positions e and g. Here, we characterized seven α3 analog polypeptides with serine (Ser), glycine (Gly), or charged residues substituted for Ala at positions e and g. The α-helix forming abilities of the substituted polypeptides were less than that of α3. The polypeptides with amino acid substitutions at position g and the polypeptide KEα3, in which Ala was substituted with charged amino acids, formed few fibrous assemblies. In contrast, polypeptides with Ala replaced by Ser at position e formed ß-sheets under several conditions. These results show that Ala residues at position e and particularly at position g are involved in the formation of fibrous assemblies.

Effects of chain length of an amphipathic polypeptide carrying the repeated amino acid sequence (LETLAKA)(n) on α-helix and fibrous assembly formation.

Polypeptide α3 (21 residues), with three repeats of a seven-amino-acid sequence (LETLAKA)(3), forms an amphipathic α-helix and a long fibrous assembly. Here, we investigated the ability of α3-series polypeptides (with 14-42 residues) of various chain lengths to form α-helices and fibrous assemblies. Polypeptide α2 (14 residues), with two same-sequence repeats, did not form an α-helix, but polypeptide α2L (15 residues; α2 with one additional leucine residue on its carboxyl terminal) did form an α-helix and fibrous assembly. Fibrous assembly formation was associated with polypeptides at least as long as polypeptide α2L and with five leucine residues, indicating that the C-terminal leucine has a critical element for stabilization of α-helix and fibril formation. In contrast, polypeptides α5 (35 residues) and α6 (42 residues) aggregated easily, although they formed α-helices. A 15-35-residue chain was required for fibrous assembly formation. Electron microscopy and X-ray fiber diffraction showed that the thinnest fibrous assemblies of polypeptides were about 20 Å and had periodicities coincident with the length of the α-helix in a longitudinal direction. These results indicated that the α-helix structures were orientated along the fibrous axis and assembled into a bundle. Furthermore, the width and length of fibrous assemblies changed with changes in the pH value, resulting in variations in the charged states of the residues. Our results suggest that the formation of fibrous assemblies of amphipathic α-helices is due to the assembly of bundles via the hydrophobic faces of the helices and extension with hydrophobic noncovalent bonds containing a leucine.

Crystallization and preliminary X-ray diffraction analysis of Bacillus subtilis YwfE, an L-amino-acid ligase.

Bacillus subtilis YwfE, an L-amino-acid ligase, catalyzes the formation of an α-dipeptide from L-amino acids in an ATP-dependent manner. In order to elucidate the substrate-recognition mode and the reaction mechanism of this ligase, native and selenomethionine-derivatized (SeMet) crystals of YwfE in the presence of ADP, MgCl(2) and the dipeptide L-Ala-L-Gln were obtained using the hanging-drop vapour-diffusion method. These crystals diffracted to 1.9 and 2.8 Å resolution, respectively. Preliminary SAD phase calculations using the data set from the SeMet crystal suggested that the crystal belonged to the hexagonal space group P6(5)22, with unit-cell parameters a = b = 90.85, c = 250.31 Å, and contained one molecule in the asymmetric unit with a solvent content of 57.3%.

Crystal structure of the AlbEF complex involved in subtilosin A biosynthesis.

Subtilosin A is a sactipeptide bacteriocin produced by Bacillus subtilis strain 168, containing intramolecular thioether bonds and a head-to-tail macrocyclic peptide bond. Macrocyclization is presumably catalyzed by AlbE and AlbF proteins encoded by the subtilosin A biosynthesis gene cluster. However, the underlying mechanism of macrocyclization remains uncertain as the tertiary structures of the proteins are undetermined. Here, we report the crystal structures of AlbE and AlbF homologs in Quasibacillus thermotolerans, wherein the subtilosin biosynthesis gene cluster is highly conserved. Structural analysis and pull-down assays revealed that AlbE and AlbF form heterodimeric complexes. Although the AlbEF complex shows structural similarity to M16B family metalloproteases, the substrate-binding chamber is shallower and more open than the other M16B family proteins. The chamber surface showed electrostatic complementarity to the precursor of subtilosin. Our findings provide insights into the role of AlbEF in metalloprotease catalysis and macrocyclic peptide bond formation.

Development of a novel peptide inhibitor of subtilisin BPN'.

Proteinaceous protease inhibitors can strongly and specifically inhibit cognate proteases, but their use as pharmaceuticals is limited by their size. As such, the development of effective protease peptide inhibitors would be beneficial for biochemical studies and drug discovery. In this study, we applied a phage display system to select subtilisin BPN'-binding peptides and evaluated their inhibitory activities against subtilisin BPN'. A 12mer peptide with an intramolecular disulfide bond inhibited subtilisin BPN' (Ki value of 13.0 nm). Further mutational analyses of the peptide resulted in the development of a short peptide inhibitor against subtilisin BPN' that showed high inhibitory activity and binding affinity (Ki value of 0.30 nm). This activity was found to be derived from the conformational rigidity caused by the intramolecular disulfide bond and the small residue at the P1' site and from the interaction of the P4 and P6' residues with subtilisin BPN'.

Increasing the hydrolysis constant of the reactive site upon introduction of an engineered Cys¹4-Cys³9 bond into the ovomucoid third domain from silver pheasant.

P14C/N39C is the disulfide variant of the ovomucoid third domain from silver pheasant (OMSVP3) introducing an engineered Cys¹4-Cys³9 bond near the reactive site on the basis of the sequence homology between OMSVP3 and ascidian trypsin inhibitor. This variant exhibits a narrower inhibitory specificity. We have examined the effects of introducing a Cys¹4-Cys³9 bond into the flexible N-terminal loop of OMSVP3 on the thermodynamics of the reactive site peptide bond hydrolysis, as well as the thermal stability of reactive site intact inhibitors. P14C/N39C can be selectively cleaved by Streptomyces griseus protease B at the reactive site of OMSVP3 to form a reactive site modified inhibitor. The conversion rate of intact to modified P14C/N39C is much faster than that for wild type under any pH condition. The pH-independent hydrolysis constant (K(hyd) °) is estimated to be approximately 5.5 for P14C/N39C, which is higher than the value of 1.6 for natural OMSVP3. The reactive site modified form of P14C/N39C is thermodynamically more stable than the intact one. Thermal denaturation experiments using intact inhibitors show that the temperature at the midpoint of unfolding at pH 2.0 is 59 °C for P14C/N39C and 58 °C for wild type. There have been no examples, except P14C/N39C, where introducing an engineered disulfide causes a significant increase in K(hyd) °, but has no effect on the thermal stability. The site-specific disulfide introduction into the flexible N-terminal loop of natural Kazal-type inhibitors would be useful to further characterize the thermodynamics of the reactive site peptide bond hydrolysis.

Distribution of dietary protein intake in daily meals influences skeletal muscle hypertrophy via the muscle clock.

The meal distribution of proteins throughout the day is usually skewed. However, its physiological implications and the effects of better protein distribution on muscle volume are largely unknown. Here, using the two-meals-per-day feeding model, we find that protein intake at the early active phase promotes overloading-induced muscle hypertrophy, in a manner dependent on the local muscle clock. Mice fed branched-chain amino acid (BCAA)-supplemented diets at the early active phase demonstrate skeletal muscle hypertrophy. However, distribution-dependent effects are not observed in ClockΔ19 or muscle-specific Bmal1 knockout mice. Additionally, we examined the relationship between the distribution of proteins in meals and muscle functions, such as skeletal muscle index and grip strength in humans. Higher muscle functions were observed in subjects who ingested dietary proteins mainly at breakfast than at dinner. These data suggest that protein intake at breakfast may be better for the maintenance of skeletal muscle mass.

Methylene Blue Inhibits Formation of Tau Fibrils but not of Granular Tau Oligomers: A Plausible Key to Understanding Failure of a Clinical Trial for Alzheimer's Disease.

Alzheimer's disease pathology is characterized by extracellular deposits of amyloid-ß (Aß) and intracellular inclusions of hyperphosphorylated tau. Although genetic studies of familial Alzheimer's disease suggest a causal link between Aß and disease symptoms, the failure of various Aß-targeted strategies to slow or halt disease progression has led to consideration of the idea that inhibition of tau aggregation might be a more promising therapeutic approach. Methylene blue (MB), which inhibits tau aggregation and rescue memory deficits in a mouse model of tauopathy, however, lacked efficacy in a recent Phase III clinical trial. In order to gain insight into this failure, the present study was designed to examine the mechanism through which MB inhibits tau aggregation. We found that MB inhibits heparin-induced tau aggregation in vitro, as measured by thioflavin T fluorescence. Further, MB reduced the amount of tau in precipitants recovered after ultracentrifugation of the aggregation mixture. Atomic force microscopy revealed that MB reduces the number of tau fibrils but increases the number of granular tau oligomers. The latter result was confirmed by sucrose gradient centrifugation: MB treatment was associated with higher levels of granular tau oligomers (fraction 3) and lower levels of tau fibrils (fractions 5 and 6). We previously demonstrated that the formation of granular tau oligomers, rather than tau fibrils, is essential for neuronal death. Thus, the fact that MB actions are limited to inhibition of tau fibril formation provides a mechanistic explanation for the poor performance of MB in the recent Phase III clinical trial.

Requirement of Ala residues at g position in heptad sequence of alpha-helix-forming peptide for formation of fibrous structure.

One feature of the alpha3-peptide, which has the amino acid sequence of (Leu-Glu-Thr-Leu-Ala-Lys-Ala)(3), that distinguishes it from many other alpha-helix-forming peptides is its ability to form fibrous assemblies that can be observed by transmission electron microscopy. In this study, the effects of Ala-->Gln substitution at the e (5th) or g (7th) position in the above heptad sequence of the alpha3-peptide on the formation of alpha-helix and fibrous assemblies were investigated by circular dichroism spectral measurement and atomic force microscopy. The 5Qalpha3-peptide obtained by Ala-->Gln substitution at the e position of the alpha3-peptide was found to form very short fibrils with long-elliptical shape, whereas the 7Qalpha3-peptide with Gln residues at the g position lost its ability to form such assemblies, in spite of alpha-helix formation in both peptides; the stabilities of both peptides decreased. These results indicate that Ala residues at the g position in the heptad sequence of the alpha3-peptide are key residues for the formation of fibrous assemblies, which may be due to hydrophobic interactions between alpha-helical bundle surfaces.

Day-Night Oscillation of Atrogin1 and Timing-Dependent Preventive Effect of Weight-Bearing on Muscle Atrophy.

BACKGROUND: Atrogin1, which is one of the key genes for the promotion of muscle atrophy, exhibits day-night variation. However, its mechanism and the role of its day-night variation are largely unknown in a muscle atrophic context. METHODS: The mice were induced a muscle atrophy by hindlimb-unloading (HU). To examine a role of circadian clock, Wild-type (WT) and Clock mutant mice were used. To test the effects of a neuronal effects, an unilateral ablation of sciatic nerve was performed in HU mice. To test a timing-dependent effects of weight-bearing, mice were released from HU for 4 h in a day at early or late active phase (W-EAP and W-LAP groups, respectively). FINDINGS: We found that the day-night oscillation of Atrogin1 expression was not observed in Clock mutant mice or in the sciatic denervated muscle. In addition, the therapeutic effects of weight-bearing were dependent on its timing with a better effect in the early active phase. INTERPRETATION: These findings suggest that the circadian clock controls the day-night oscillation of Atrogin1 expression and the therapeutic effects of weight-bearing are dependent on its timing. FUND: Council for Science, Technology, and Innovation, SIP, "Technologies for creating next-generation agriculture, forestry, and fisheries".

Effects of hydrophobic amino acid substitution in Pleurotus ostreatus proteinase A inhibitor 1 on its structure and functions as protease inhibitor and intramolecular chaperone.

We previously demonstrated that Pleurotus ostreatus proteinase A inhibitor 1 (POIA1) could function as an intramolecular chaperone of subtilisin BPN', as in the case of the propeptide of subtilisin BPN', and that its Phe44 --> Ala mutant, which lost its tertiary structure, could not assist the refolding of subtilisin BPN'. In this study, we examined the effects of hydrophobic amino acid substitutions at other sites and substitutions of Phe44 with other hydrophobic residues on the structure and functions of POIA1. These mutations were introduced into POIA1cm that had been obtained by the substitution of the C-terminal six residues of POIA1 with those of the propeptide of subtilisin BPN'. When Ile32 or Ile64 was substituted with Ala, the tertiary structure of the resultant mutant was markedly destroyed, and the activities as a protease inhibitor and an intramolecular chaperone were significantly lowered. Among the position 44 mutants, the Phe44 --> Val mutant was a much less effective intramolecular chaperone with conversion to a digestible inhibitor, possibly owing to destruction of the tertiary structure. On the other hand, the Phe44 --> Leu or Ile mutant maintained its tertiary structure, and hence could function as a more effective intramolecular chaperone than the Phe44 --> Val mutant. Furthermore, since the Phe44 --> Leu mutant was a more susceptible inhibitor than POIA1cm, the halo formed around a colony of Bacillus cells transformed with a plasmid encoding this mutant was larger than others. These results clearly show the close relationship between the tertiary structure and functions of POIA1 as a protease inhibitor and an intramolecular chaperone, and that a combination of such inhibitory properties and intramolecular chaperone activity of POIA1 might affect the diameter of the halo formed around Bacillus colonies in vivo.

Investigation of 5-HT3 receptor-triggered serotonin release from guinea-pig isolated colonic mucosa: a role of PYY-containing endocrine cell.

The effect of a 5-HT3 receptor-selective agonist SR57227A was investigated on the outflow of 5-hydroxytryptamine (5-HT) from isolated muscle layer-free mucosal preparations of guinea-pig colon. The mucosal preparations were incubated in vitro and the outflow of 5-HT from these preparations was determined by high-performance liquid chromatography with electrochemical detection. SR57227A (100µM) produced a tetrodotoxin-resistant and sustained increase in the outflow of 5-HT from the mucosal preparations. The SR57227A-evoked sustained 5-HT outflow was completely inhibited by the 5-HT3 receptor antagonist ramosetron (1µM). The neuropeptide Y1 receptor antagonist BIBO3304 (100nM) partially inhibited the SR57227A-evoked sustained 5-HT outflow, but the Y2 receptor antagonist BIIE0246 (1µM) or the glucagon-like peptide-1 (GLP-1) receptor antagonist exendin-(9-39) (1µM), showed a minimal effect on the SR57227A-evoked sustained 5-HT outflow. In the presence of BIBO3304 (100nM) and exendin-(9-39) (1µM), SR57227A (100µM) failed to produce a sustained increase in the outflow of 5-HT. The Y1 receptor agonist [Leu31, Pro34]-neuropeptide Y (10nM), but not GLP-1-(7-36) amide (100nM), produced a sustained increase in the outflow of 5-HT. We found that 5-HT3 receptor-triggered 5-HT release from guinea-pig colonic mucosa is mediated by the activation of 5-HT3 receptors located at endocrine cells (enterochromaffin cells and peptide YY (PYY)-containing endocrine cells). The activation of both Y1 and GLP-1 receptors appears to be required for the maintenance of 5-HT3 receptor-triggered 5-HT release. It is therefore considered that 5-HT3 receptors located at colonic mucosa play a crucial role in paracrine signaling between enterochromaffin cells and PYY-containing endocrine cells.

The structure of the third intracellular loop of the muscarinic acetylcholine receptor M2 subtype.

We have examined whether the long third intracellular loop (i3) of the muscarinic acetylcholine receptor M2 subtype has a rigid structure. Circular dichroism (CD) and nuclear magnetic resonance spectra of M2i3 expressed in and purified from Escherichia coli indicated that M2i3 consists mostly of random coil. In addition, the differential CD spectrum between the M2 and M2deltai3 receptors, the latter of which lacks most of i3 except N- and C-terminal ends, gave no indication of secondary structure. These results suggest that the central part of i3 of the M2 receptor has a flexible structure.

The effects of the side chains of hydrophobic aliphatic amino acid residues in an amphipathic polypeptide on the formation of alpha helix and its association.

The polypeptide alpha3, which was synthesized by us to produce an amphipathic helix structure, contains the regular three times repeated sequence (LETLAKA)(3), and alpha3 forms a fibrous assembly. To clarify how the side chains of amino acid residues affect the formation of alpha helix, Leu residues, which are located in the hydrophobic surface of an amphipathic helix, were replaced by other hydrophobic aliphatic amino acid residues systematically, and the characters of the resulting polypeptides were studied. According to the circular dichroism (CD) spectra, the Ile-substituted polypeptides formed alpha helix like alpha3. However, their helix formation ability was weaker than that of alpha3 under some conditions. The Val-substituted polypeptides formed alpha helix only under restricted condition. The Ala-substituted polypeptides did not form alpha helix under any condition. Thus, it is clear that the order of the alpha helix formation ability is as follows: Leu >or= Ile > Val > Ala. The formation of alpha helix was confirmed by Fourier Transform Infrared (FTIR) spectra. Through electron microscopic observation, it was clarified that the formation of the alpha helix structure correlates with the formation of a fibrous assembly. The amphipathic alpha helix structure would be stabilized by the formation of the fibrous assembly.

The suppressant effect of GEA3162 on spontaneous serotonin release from human colonic mucosa in vitro.

The effect of a lipophilic nitric oxide (NO)-releasing compound 5-amino-3-(3,4-dichlorophenyl) 1,2,3,4-oxatriazolium (GEA3162) on the spontaneous release of 5-hydroxytryptamine (5-HT) from human colonic mucosa was investigated in vitro. In the presence of tetrodotoxin, spontaneous outflow of 5-HT from the human colonic mucosa was measured by high-performance liquid chromatography with electrochemical detection. GEA3162 concentration-dependently suppressed the 5-HT outflow, but neither the NO-activated soluble guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazolo[4,3-a]quinoxalin-1-one (ODQ) nor peroxynitrite scavenger ebselen affected the suppressant effect of GEA3162. Moreover, neither the L-type calcium channel blocker nicardipine, NO synthase inhibitor l-N(G)-nitroarginine methyl ester nor guanylate cyclase activator guanylin affected the spontaneous 5-HT outflow. These results indicate that human colonic mucosa is capable of eliciting tetrodotoxin-resistant and nicardipine-insensitive 5-HT release, and that GEA3162 can suppress the 5-HT release via an action on colonic mucosa through mechanism independent of ODQ-sensitive cyclic GMP system or peroxynitrite generation.

Cloning and expression of the enolase gene from Desulfovibrio vulgaris (Miyazaki F).

The gene encoding an enolase from Desulfovibrio vulgaris (Miyazaki F) was cloned and overexpressed in Escherichia coli. A 2.1-kb DNA fragment, isolated from D. vulgaris (Miyazaki F) by double digestion with PstI and BamHI, contained an enolase gene (eno) and part of the methylenetetrahydrofolate dehydrogenase gene (folD). The nucleotide sequence of eno indicates that the protein monomer is composed of 434 amino acids. An expression system for eno under control of the T7 promoter was constructed in E. coli. The purified His-tagged enolase formed a homooctamer and was active in the formation of phosphoenolpyruvate (PEP) as well as in the reverse reaction, the formation of D-(+)-2-phosphoglyceric acid (2-PGA). The pH dependence and kinetic properties of the recombinant enolase from the sulfate-reducing bacterium were also studied. The amounts of eno mRNA when the bacterium was grown on glycerol or glucose were compared to that when D. vulgaris was grown on lactate.

Inhibitor-assisted refolding of protease: a protease inhibitor as an intramolecular chaperone.

Pleurotus ostrearus proteinase A inhibitor 1 (POIA1), which was discovered as a protease inhibitor, is unique in that it shows sequence homology to the propeptide of subtilisin, which functions as an intramolecular of a cognate protease. In this study, we demonstrate that POIA1 can function as an intramolecular chaperone of subtilisin by in vitro and in vivo experiments. The specific cleavage between POIA1 and the mature region of subtilisin BPN' occurred in a refolding process of a chimera protein, and Bacillus cells transformed with a chimera gene formed a halo on a skim milk plate. The mutational analyses of POIA1 in the chimera protein suggested that the tertiary structure of POIA1 is required for such a function, and that an increase in its ability to bind to subtilisin BPN' makes POIA1 a more effective intramolecular chaperone.

Structure of POIA1, a homologous protein to the propeptide of subtilisin: implication for protein foldability and the function as an intramolecular chaperone.

Solution structure of POIA1 (Pleurotus ostreatus proteinase A inhibitor 1), which functions as an intramolecular chaperone and as an inhibitor to subtilisin, was determined. By making use of the fact that POIA1 is the only structured protein that shows homology to the propeptide of subtilisin, which is unstructured by itself, foldability of this protein was elucidated. It became clear that the evolutionarily conserved residues play two important roles, one for the maintenance of its own structure, and the other for the interaction with subtilisin. Structural softness and mutational tolerance contained in the POIA1 structure makes it an ideal material for designing a foldable protein.

An Endogenous Tachykinergic NK2/NK3 Receptor Cascade System Controlling the Release of Serotonin from Colonic Mucosa.

5-Hydroxytryptamine (5-HT) released from colonic mucosal enterochromaffin (EC) cells is a major signaling molecule, which participates in the pathophysiological regulation of colonic functions in gut disorder including irritable bowel syndrome (IBS), but the endogenous modulator system for the 5-HT release is not yet well elucidated. Our in vitro studies in guinea-pig colon have indicated that the cascade pathway of neuronal tachykinergic NK3 receptors and NK2 receptors on peptide YY (PYY)-containing endocrine L cells represents an endogenous modulator system for 5-HT release from EC cells and that melatonin, endogenous tachykinins and PYY play important roles in modulation of the release of 5-HT from EC cells via the endogenous NK2/NK3 receptor cascade system. This review aims at examining the potential role of the endogenous tachykinergic NK2/NK3 receptor cascade system controlling the release of 5-HT from EC cells, with special attention being paid to the pathophysiology of gut disorders including IBS.