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A novel CCK-dependent vasorelaxing RF decreases both blood pressure and food intake.

Poly-L-␣-amino acids have various applications because of their biodegradable properties and biocompatibility. Microorganisms contain several enzymes that catalyze the polymerization of L-amino acids in an ATP-dependent manner, but the products from these reactions contain amide linkages at the side residues of amino acids: e.g., poly-␥-glutamic acid, poly--lysine, and cyanophycin. In this study, we found a novel catalytic activity of RimK, a ribosomal protein S6-modifying enzyme derived from Escherichia coli K-12. This enzyme catalyzed poly-␣-glutamic acid synthesis from unprotected L-glutamic acid (Glu) by hydrolyzing ATP to ADP and phosphate. RimK synthesized poly-␣-glutamic acid of various lengths; matrix-assisted laser desorption ionization-time of flight-mass spectrometry showed that a 46-mer of Glu (maximum length) was synthesized at pH 9. Interestingly, the lengths of polymers changed with changing pH. RimK also exhibited 86% activity after incubation at 55°C for 15 min, thus showing thermal stability. Furthermore, peptide elongation seemed to be catalyzed at the C terminus in a stepwise manner. Although RimK showed strict substrate specificity toward Glu, it also used, to a small extent, other amino acids as C-terminal substrates and synthesized heteropeptides. In addition, RimK-catalyzed modification of ribosomal protein S6 was confirmed. The number of Glu residues added to the protein varied with pH and was largest at pH 9.5.Poly-L-amino acids possess biodegradable properties and are therefore useful in various fields, including food science, medicine, and cosmetics. Polyaspartic acid is used as a biodegradable substitute for synthetic polyacrylate (25), and poly-␣-glutamic acid finds application in a wide variety of surgical and pharmaceutical products (e.g., for enhancement of solubility and control of half-life of drugs) (27). Furthermore, L-arginine (Arg)-rich peptides, which can permeate cell membranes, are used for intracellular delivery of macromolecules (7). In addition, unique polyamino acids produced by various microorganisms, including poly-␥-glutamic acid, poly-ε-lysine, and cyanophycin (multi-L-arginyl-poly[L-aspartic acid]), have been well studied and widely applied (6,21,26). These acids have a ␥-, ε-, or ␤-amide linkage. Both poly-␥-glutamic acid and poly-ε-lysine have characteristic abilities of high water absorbency and antimicrobial activity, respectively, and are therefore used on a commercial scale. Previous studies have revealed the biosynthetic mechanisms of these polyamino acids and have achieved their mass production (5, 33). In contrast, poly-L-amino acids with only an ␣-amide linkage have not been found in microorganisms, probably because poly-L-␣-amino acids might be hydrolyzed by proteases and peptidases contained in the microorganisms. Therefore, poly-L-␣-amino acids may not be detectable even in organisms containing an enzyme possessing poly-L-␣-amino acid-synthesizing activity.To create a supply of useful poly-␣-amino acids, their potential production by chemical and en...

Functional peptides are expected to be beneficial compounds that improve our quality of life. To address the growing need for functional peptides, we have examined peptide synthesis by using microbial enzymes. L-Amino acid ligase (Lal) catalyzes the condensation of unprotected amino acids in an ATP-dependent manner and is applicable to fermentative production. Hence, Lal is a promising enzyme to achieve cost-effective synthesis. To obtain a Lal with novel substrate specificity, we focused on the putative Lal involved in the biosynthesis of the dipeptidic phytotoxin designated tabtoxin. The tabS gene was cloned from Pseudomonas syringae NBRC14081 and overexpressed in Escherichia coli cells. The recombinant TabS protein produced showed the broadest substrate specificity of any known Lal; it detected 136 of 231 combinations of amino acid substrates when dipeptide synthesis was examined. In addition, some new substrate specificities were identified and unusual amino acids, e.g., L-pipecolic acid, hydroxy-L-proline, and ␤-alanine, were found to be acceptable substrates. Furthermore, kinetic analysis and monitoring of the reactions over a short time revealed that TabS showed distinct substrate selectivity at the N and C termini, which made it possible to specifically synthesize a peptide without by-products such as homopeptides and heteropeptides with the reverse sequence. TabS specifically synthesized the following functional peptides, including their precursors: L-arginyl-L-phenylalanine (antihypertensive effect; yield, 62%), L-leucyl-L-isoleucine (antidepressive effect; yield, 77%), L-glutaminyl-L-tryptophan (precursor of L-glutamyl-L-tryptophan, which has antiangiogenic activity; yield, 54%), L-leucyl-L-serine (enhances saltiness; yield, 83%), and L-glutaminyl-L-threonine (precursor of L-glutamyl-L-threonine, which enhances saltiness; yield, 96%). Furthermore, our results also provide new insights into tabtoxin biosynthesis.

Muscle fiber contraction involves chemical reactions and structural changes that are sensitive to temperature. Ishii et al. show that rapid heating causes cardiac thin filament sliding, and the data could indicate that thin filaments are partially activated during diastole at mammalian body temperature.