Masaki Kamakura

Royalactin induces queen differentiation in honeybees

The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway.

Storage-dependent Degradation of 57-kDa Protein in Royal Jelly: a Possible Marker for Freshness

In order to find a maker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40°C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4°C to 50°C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.

Identification of the amino acid residue of CYP27B1 responsible for binding of 25-hydroxyvitamin D3 whose mutation causes vitamin D-dependent rickets type 1.

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1α-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1α-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.

Structure-function analysis of vitamin D 24-hydroxylase (CYP24A1) by site-directed mutagenesis: amino acid residues responsible for species-based difference of CYP24A1 between humans and rats.

Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1α,25-dihydroxyvitamin D3 [1α,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1α,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.

Metabolism of sesamin by cytochrome P450 in human liver microsomes.

Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest kcat/Km value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Structure-based design of a highly active vitamin D hydroxylase from Streptomyces griseolus CYP105A1

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

Bioconversion of vitamin D to its active form by bacterial or mammalian cytochrome P450

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in the bioconversion process that produces 1α,25-dihydroxyvitamin D₃. In addition, the directed evolution study of CYP107 has significantly enhanced its activity. On the other hand, we found that Streptomyces griseolus CYP105A1 can convert vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity. To date, multiple vitamin D hydroxylases have been found in bacteria, fungi, and mammals, suggesting that vitamin D is a popular substrate of the enzymes belonging to the P450 superfamily. A combination of these cytochrome P450s would produce a large number of compounds from vitamin D and its analogs. Therefore, we believe that the bioconversion of vitamin D and its analogs is one of the most promising P450 reactions in terms of practical application.

Anti-proliferative activity of 25-hydroxyvitamin D3 in human prostate cells.

1α-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). First, we measured the cell-derived 1α,25(OH)2D3 level in immortalized human prostate cell (PZ-HPV-7) using [(3)H]-25(OH)D3. The effects of the cell-derived 1α,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects, whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts its biological functions. DNA microarray and real-time RT-PCR analyses suggest that semaphorin 3B, cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3.

Three-step hydroxylation of vitamin D3 by a genetically engineered CYP105A1

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D3 to its biologically active form, 1α,25‐dihydroxyvitamin D3 [1α,25(OH)2D3], suggesting the possibility for R73V/R84A to produce 1α,25(OH)2D3. Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25‐hydroxyvitamin D3 [25(OH)D3] and 1α,25(OH)2D3 were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)2D3, the second‐step metabolite of vitamin D3, was observed, although a considerable amount of vitamin D3 still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)3D3 and 1α,25(S),26(OH)3D3, both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5 : 1. The crystal structure of the double variant with 1α,25(OH)2D3 and a docking model of 1α,25(OH)2D3 in its active site strongly suggest a hydrogen‐bond network including the 1α‐hydroxyl group, and several water molecules play an important role in the substrate‐binding for 26‐hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D3 to produce biologically useful compounds.

Metabolism of 1α,25-dihydroxyvitamin D2 by human CYP24A1

The metabolism of 1alpha,25-dihydroxyvitamin D2 (1alpha,25(OH)2D2) by human CYP24A1 was examined using the recombinant enzyme expressed in Escherichia coli cells. HPLC analysis revealed that human CYP24A1 produces at least 10 metabolites, while rat CYP24A1 produces only three metabolites, indicating a remarkable species-based difference in the CYP24A1-dependent metabolism of 1alpha,25(OH)2D2 between humans and rats. LC-MS analysis and periodate treatment of the metabolites strongly suggest that human CYP24A1 converts 1alpha,25(OH)2D2 to 1alpha,24,25,26(OH)4D2, 1alpha,24,25,28(OH)4D2, and 24-oxo-25,26,27-trinor-1alpha(OH)D2 via 1alpha,24,25(OH)3D2. These results indicate that human CYP24A1 catalyzes the C24-C25 bond cleavage of 1alpha,24,25(OH)2D2, which is quite effective in the inactivation of the active form of vitamin D2. The combination of hydroxylation at multiple sites and C-C bond cleavage could form a large number of metabolites. Our findings appear to be useful to predict the metabolism of vitamin D2 and its analogs in the human body.