Diversity and evolution of serotonergic cells in taste buds of elasmobranchs and ancestral actinopterygian fish.

A subset of gustatory cells are serotonin immunoreactive (ir) in the mammalian taste bud. In the taste bud of lamprey, elongated gustatory-like cells are also serotonin-ir. In contrast, flattened serotonin-ir cells are located only in the basal region of the taste buds in the teleosts and amphibians. These serotonin-ir cells are termed as basal cells. To evaluate the evolution and diversity of serotonergic cells in the taste bud of amniote animals, we explored the distribution and morphology of serotonin-ir cells in the taste buds of ancestral actinopterygian fish (spotted gar, sturgeon, Polypterus senegalus) and elasmobranch (stingray). In all examined animals, the taste buds contained serotonin-ir cells in their basal part. The number of serotonin-ir basal cells in each taste bud was different between these fish species. They were highest in the stingray and decreased in the order of the Polypterus, sturgeon, and gar. While serotonin immunoreactivity was observed only in the basal cells in the taste buds of the ancestral actinopterygian fish, some elongated cells were also serotonin-ir in addition to the basal cells in the stingray taste buds. mRNA of tryptophan hydroxylase 1 (tph1), a rate-limiting enzyme of the serotonin synthesis, is expressed in both the elongated and basal cells of stingray taste buds, indicating that these cells synthesize the serotonin by themselves. These results suggest that the serotonin-ir basal cells arose from the ancestor of the cartilaginous fish, and serotonin-ir cells in the elasmobranch taste bud exhibit an intermediate aspect between the lamprey and actinopterygian fish.

Morphological analysis of the cerebellum and its efferent system in a basal actinopterygian fish, Polypterus senegalus.

Although all vertebrate cerebella contain granule cells, Purkinje cells, and efferent neurons, the cellular arrangement and neural circuitry are highly diverse. In amniotes, cerebellar efferent neurons form clusters, deep cerebellar nuclei, lie deep in the cerebellum, and receive synaptic inputs from Purkinje cells but not granule cells. However, in the cerebellum of teleosts, the efferent neurons, called eurydendroid cells, lie near the cell bodies of Purkinje cells and receive inputs both from axons of Purkinje cells and granule cell parallel fibers. It is largely unknown how the cerebellar structure evolved in ray-finned fish (actinopterygians). To address this issue, we analyzed the cerebellum of a bichir Polypterus senegalus, one of the most basal actinopterygians. We found that the cell bodies of Purkinje cells are not aligned in a layer; incoming climbing fibers terminate mainly on the basal portion of Purkinje cells, revealing that the Polypterus cerebellum has unique features among vertebrate cerebella. Retrograde labeling and marker analyses of the efferent neurons revealed that their cell bodies lie in restricted granular areas but not as deep cerebellar nuclei in the cerebellar white matter. The efferent neurons have long dendrites like eurydendroid cells, although they do not reach the molecular layer. Our findings suggest that the efferent system of the bichir cerebellum has intermediate features between teleosts and amniote vertebrates, and provides a model to understand the basis generating diversity in actinopterygian cerebella.

Structural basis of the correct subunit assembly, aggregation, and intracellular degradation of nylon hydrolase.

Nylon hydrolase (NylC) is initially expressed as an inactive precursor (36 kDa). The precursor is cleaved autocatalytically at Asn266/Thr267 to generate an active enzyme composed of an α subunit (27 kDa) and a ß subunit (9 kDa). Four αß heterodimers (molecules A-D) form a doughnut-shaped quaternary structure. In this study, the thermostability of the parental NylC was altered by amino acid substitutions located at the A/D interface (D122G/H130Y/D36A/L137A) or the A/B interface (E263Q) and spanned a range of 47 °C. Considering structural, biophysical, and biochemical analyses, we discuss the structural basis of the stability of nylon hydrolase. From the analytical centrifugation data obtained regarding the various mutant enzymes, we conclude that the assembly of the monomeric units is dynamically altered by the mutations. Finally, we propose a model that can predict whether the fate of the nascent polypeptide will be correct subunit assembly, inappropriate protein-protein interactions causing aggregation, or intracellular degradation of the polypeptide.

IgY-binding peptide screened from a random peptide library as a ligand for IgY purification.

Chicken egg yolk immunoglobulin (IgY) is a functional substitute for mammalian IgG for antigen detection. Traditional IgY purification methods involve multi-step procedures resulting in low purity and recovery of IgY. In this study, we developed a simple IgY purification system using IgY-specific peptides identified by T7 phage display technology. From disulfide-constrained random peptide libraries constructed on a T7 phage, we identified three specific binding clones (Y4-4, Y5-14, and Y5-55) through repeated biopanning. The synthetic peptides showed high binding specificity to IgY-Fc and moderate affinity for IgY-Fc (Kd : Y4-4 = 7.3 ± 0.2 µM and Y5-55 = 4.4 ± 0.1 µM) by surface plasmon resonance analysis. To evaluate the ability to purify IgY, we performed immunoprecipitation and affinity high-performance liquid chromatography using IgY-binding peptides; the result indicated that these peptides can be used as affinity ligands for IgY purification. We then used a peptide-conjugated column to purify IgY from egg yolks pre-treated using an optimized delipidation technique. Here, we report the construction of a cost-effective, one-step IgY purification system, with high purity and recovery. © 2017 The Authors. Journal of Peptide Science published by European Peptide Society and John Wiley & Sons Ltd.

Biosynthesis-inspired deracemizative production of d-luciferin by combining luciferase and thioesterase.

Due to the strict enantioselectivity of firefly luciferase, only d-luciferin can be used as a substrate for bioluminescence reactions. Unfortunately, luciferin racemizes easily and accumulation of nonluminous l-luciferin has negative influences on the light emitting reaction. Thus, maintaining the enantiopurity of luciferin in the reaction mixture is one of the most important demands in bioluminescence applications using firefly luciferase. In fireflies, however, l-luciferin is the biosynthetic precursor of d-luciferin, which is produced from the L-form undergoing deracemization. This deracemization consists of three successive reactions: l-enantioselective thioesterification by luciferase, in situ epimerization, and hydrolysis by thioesterase. In this work, we introduce a deracemizative luminescence system inspired by the biosynthetic pathway of d-luciferin using a combination of firefly luciferase from Luciola cruciata (LUC-G) and fatty acyl-CoA thioesterase II from Escherichia coli (TESB). The enzymatic reaction property analysis indicated the importance of the concentration balance between LUC-G and TESB for efficient d-luciferin production and light emission. Using this deracemizative luminescence system, a highly sensitive quantitative analysis method for l-cysteine was constructed. This LUC-G-TESB combination system can improve bioanalysis applications using the firefly bioluminescence reaction by efficient deracemization of D-luciferin.

Purification and gene cloning of an enantioselective thioesterification enzyme from Brevibacterium ketoglutamicum KU1073, a deracemization bacterium of 2-(4-chlorophenoxy)propanoic acid.

We succeeded in the purification and gene cloning of a new enzyme, α-methyl carboxylic acid deracemizing enzyme 1 (MCAD1) from Brevibacterium ketoglutamicum KU1073, which catalyzes the (S)-enantioselective thioesterification reaction of 2-(4-chlorophenoxy)propanoic acid (CPPA). The cloned gene of MCAD1 contained an ORF of 1,623 bp, encoding a polypeptide of 540 amino acids. In combination with cofactors ATP, coenzyme A (CoASH), and Mg(2+), MCAD1 demonstrated perfect enantioselectivity toward CPPA. The optimal pH and temperature for reaction were found to be 7.25 and 30 °C. Under these conditions, the K(m) and k(cat) values for (S)-CPPA were 0.92 ± 0.17 mM and 0.28 ± 0.026 s(-1) respectively. The results for substrate specificity revealed that MCAD1 had highest activity toward fatty acid tails with a medium chain-length (C(8)). This result indicates that MCAD1 should be classified into a family of medium-chain acyl-CoA synthetase. This novel activity has never been reported for this family.

X-ray crystallographic analysis of the 6-aminohexanoate cyclic dimer hydrolase: catalytic mechanism and evolution of an enzyme responsible for nylon-6 byproduct degradation.

We performed x-ray crystallographic analyses of the 6-aminohexanoate cyclic dimer (Acd) hydrolase (NylA) from Arthrobacter sp., an enzyme responsible for the degradation of the nylon-6 industry byproduct. The fold adopted by the 472-amino acid polypeptide generated a compact mixed alpha/beta fold, typically found in the amidase signature superfamily; this fold was especially similar to the fold of glutamyl-tRNA(Gln) amidotransferase subunit A (z score, 49.4) and malonamidase E2 (z score, 44.8). Irrespective of the high degree of structural similarity to the typical amidase signature superfamily enzymes, the specific activity of NylA for glutamine, malonamide, and indoleacetamide was found to be lower than 0.5% of that for Acd. However, NylA possessed carboxylesterase activity nearly equivalent to the Acd hydrolytic activity. Structural analysis of the inactive complex between the activity-deficient S174A mutant of NylA and Acd, performed at 1.8 A resolution, suggested the following enzyme/substrate interactions: a Ser(174)-cis-Ser(150)-Lys(72) triad constitutes the catalytic center; the backbone N in Ala(171) and Ala(172) are involved in oxyanion stabilization; Cys(316)-S(gamma) forms a hydrogen bond with nitrogen (Acd-N(7)) at the uncleaved amide bond in two equivalent amide bonds of Acd. A single S174A, S150A, or K72A substitution in NylA by site-directed mutagenesis decreased the Acd hydrolytic and esterolytic activities to undetectable levels, indicating that Ser(174)-cis-Ser(150)-Lys(72) is essential for catalysis. In contrast, substitutions at position 316 specifically affected Acd hydrolytic activity, suggesting that Cys(316) is responsible for Acd binding. On the basis of the structure and functional analysis, we discussed the catalytic mechanisms and evolution of NylA in comparison with other Ser-reactive hydrolases.