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Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum.

Nucleotide binding domain and leucine-rich repeat (NLR)-containing family proteins function as intracellular immune sensors in both plants and animals. In plants, the downstream components activated by NLR family proteins and the immune response mechanisms induced by these downstream molecules are largely unknown. We have previously found that the small GTPase OsRac1, which acts as a molecular switch in rice immunity, is activated by Pit, an NLR-type resistance (R) protein to rice blast fungus, and this activation plays critical roles in Pit-mediated immunity. However, the sites and mechanisms of activation of Pit in vivo remain unknown. To clarify the mechanisms involved in the localization of Pit, we searched for consensus sequences in Pit that specify membrane localization and found a pair of potential palmitoylation sites in the N-terminal coiled-coil region. Although wild-type Pit was localized mainly to the plasma membrane, this membrane localization was compromised in a palmitoylation-deficient mutant of Pit. The palmitoylation-deficient Pit displayed significantly lower affinity for OsRac1 on the plasma membrane, thereby resulting in failures of the Pit-mediated cell death, the production of reactive oxygen species, and disease resistance to rice blast fungus. These results indicate that palmitoylation-dependent membrane localization of Pit is required for the interaction with and the activation of OsRac1 and that OsRac1 activation by Pit is vital for Pit-mediated disease resistance to rice blast fungus.

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.

SummaryThe nucleotide-binding domain and leucine-rich repeat-containing (NLR) family proteins recognize pathogen-derived molecules and trigger immune responses in both plants and animals. In plants, the direct or indirect recognition of specific pathogen effectors by NLRs culminates in a hypersensitive response (HR) and the production of reactive oxygen species (ROS), key components of the plant defense response. However, the molecules activated by NLRs and how they induce immune responses are largely unknown. We found that the rice GTPase OsRac1 at the plasma membrane interacts directly with Pit, an NLR protein that confers resistance to the rice blast fungus. OsRac1 contributes to Pit-mediated ROS production as well as the HR and is required for Pit-mediated disease resistance in rice. Furthermore, the active form of Pit induces the activation of OsRac1 at the plasma membrane. Thus, OsRac1 is activated by Pit during pathogen attack and plays a critical role in Pit-mediated immunity in rice.

Objective The purpose of this study was to assess whether laughter influences the expression of the receptor gene for prorenin that participates in the progression of diabetic nephropathy. Methods Sixteen normal subjects and 23 patients with type 2 diabetes [12 nephropathy (−) and 11 nephropathy (+)] were recruited to examine the effects of laughter on the modulation of prorenin receptor gene expression. After watching a comedy show, laughter-induced changes in the levels of blood prorenin and the expression of prorenin receptor gene were analyzed by an antibody-activating direct enzyme kinetic assay and by reverse transcriptase polymerase chain reaction, respectively. Results In diabetic patients, laughter decreased the level of blood prorenin [93.4–60.4 ng/l in nephropathy (−) patients, 196.6–166.7 ng/l in nephropathy (+) patients] and up-regulated the prorenin receptor gene [1.49-fold in nephropathy (−) patients, 1.46-fold in nephropathy (+) patients]. No significant changes in the expression of this gene were recognized in normal subjects. Conclusion The beneficial effects of laughter on preventing the exacerbation of diabetic nephropathy are strongly suggested in terms of normalizing the expression of the prorenin receptor gene followed by reducing the level of blood prorenin.

Purpose To report a case of optic neuropathy associated with prolonged use of a new antibiotic, linezolid. Design Interventional case report. Methods Optic and peripheral neuropathy occurred in a 27-year-old woman who was undergoing prolonged linezolid therapy for osteomyelitis. The use of corticosteroid exacerbated visual loss. Discontinuation of the linezolid therapy resulted in marked improvement of optic neuropathy. Results Linezolid-induced optic neuropathy was diagnosed in this case, because prolonged linezolid therapy was performed at a higher dose than usual and because the patient recovered after linezolid therapy was discontinued. Conclusions With prolonged linezolid therapy, ophthalmologists should be aware that bilateral optic neuropathy can occur and that corticosteroids are not indicated for this neuropathy.

An in vitro assay of human sperm function was devised to evaluate two initial stages of sperm-ovum interaction: penetration through the zona pellucida and sperm entry into the ooplasm. The former stage was assessed with immature human oocytes and the latter with mature zona-free hamster eggs. The two gamete populations were incubated together in the same suspension of capacitated spermatozoa. Spermatozoa from five of six fertile donors were able to penetrate through the human zona pellucida into the perivitelline space, and to enter the zona-free hamster egg. Spermatozoa from one fertile donor consistently penetrated the zona pellucida, but fertilized only 1 of 44 hamster eggs. Several types of sperm dysfunction during gamete interaction were observed in a group of 21 patients with suspected infertility: (1) failure to bind to the zona pellucida, (2) zona binding with failure to penetrate, (3) incomplete zona penetration, and (4) zona penetration with poor sperm entry into the ooplasm. Eleven of the twelve cases of apparent fertilization dysfunction would have been detected by the human oocyte assay alone, and eight of the twelve would have been diagnosed by incubation with only the zona-free hamster eggs. The success of gamete interaction in vitro could not be correlated with any of the classic parameters of semen quality. Some men with "normal" semen and unexplained infertility had apparent dysfunction of gamete interaction, while others gave positive results. These experiments indicate that, at the present time, in vitro tests of gamete interaction should be reserved for selected cases of unexplained infertility. In the future they may prove useful in identifying couples with male infertility as candidates for in vitro fertilization and embryo transfer.