[Research progress in biosynthesis-related enzymes of coumarin compounds and their bioactivities].

Coumarins are natural products with benzopyran ring as the parent nucleus. Numerous coumarin derivatives exhibit a variety of pharmacological activities, including antibacterial, anti-inflammatory, antitumor, anti-coagulant, anti-osteoporotic, and insecticidal activities. Therefore, they play an important role in both medicine and agriculture. The development and utilization of coumarin derivatives have attracted increasing attention. The advancement of gene sequencing technology and the rapid progress in synthetic bio-logy have led to significant advancement in the biosynthesis of coumarin derivatives, and has received increasing attention from global researchers. This paper presents a comprehensive overview of the key biosynthesis-related enzymes of coumarin derivatives, such as cytochrome P450 enzyme(CYP450), prenyltransferase(PT), UDP-glucosyltransferase(UGT). Additionally, the pharmacological activities of these enzymes, including anti-tumor, anti-inflammatory, antioxidant, and antibacterial activities, are systematically summarized. This review aims to provide a valuable reference for the biosynthesis of coumarin derivatives and further exploration of their medicinal potential.

Prenylation of aromatic amino acids and plant phenolics by an aromatic prenyltransferase from Rasamsonia emersonii.

Dimethylallyl tryptophan synthases (DMATSs) are aromatic prenyltransferases that catalyze the transfer of a prenyl moiety from a donor to an aromatic acceptor during the biosynthesis of microbial secondary metabolites. Due to their broad substrate scope, DMATSs are anticipated as biotechnological tools for producing bioactive prenylated aromatic compounds. Our study explored the substrate scope and product profile of a recombinant RePT, a novel DMATS from the thermophilic fungus Rasamsonia emersonii. Among a variety of aromatic substrates, RePT showed the highest substrate conversion for L-tryptophan and L-tyrosine (> 90%), yielding two mono-prenylated products in both cases. Nine phenolics from diverse phenolic subclasses were notably converted (> 10%), of which the stilbenes oxyresveratrol, piceatannol, pinostilbene, and resveratrol were the best acceptors (37-55% conversion). The position of prenylation was determined using NMR spectroscopy or annotated using MS2 fragmentation patterns, demonstrating that RePT mainly catalyzed mono-O-prenylation on the hydroxylated aromatic substrates. On L-tryptophan, a non-hydroxylated substrate, it preferentially catalyzed C7 prenylation with reverse N1 prenylation as a secondary reaction. Moreover, RePT also possessed substrate-dependent organic solvent tolerance in the presence of 20% (v/v) methanol or DMSO, where a significant conversion (> 90%) was maintained. Our study demonstrates the potential of RePT as a biocatalyst for the production of bioactive prenylated aromatic amino acids, stilbenes, and various phenolic compounds. KEY POINTS: • RePT catalyzes prenylation of diverse aromatic substrates. • RePT enables O-prenylation of phenolics, especially stilbenes. • The novel RePT remains active in 20% methanol or DMSO.

Coupling the Isopentenol Utilization Pathway and Prenyltransferase for the Biosynthesis of Licoflavanone in Recombinant Escherichia coli.

Prenylflavonoids are promising candidates for food additives and functional foods due to their diverse biological activities and potential health benefits. However, natural prenylflavonoids are generally present in low abundance and are limited to specific plant species. Here, we report the biosynthesis of licoflavanone from naringenin and prenol by recombinant Escherichia coli. By investigating the activities of seven different sources of prenyltransferases overexpressed in E. coli toward various flavonoid substrates, the prenyltransferase AnaPT exhibits substrate preference when naringenin serves as the prenyl acceptor. Furthermore, licoflavanone production was successfully achieved by coupling the isopentenol utilization pathway and AnaPT in recombinant E. coli. In addition, the effects of fermentation temperatures, induction temperatures, naringenin concentrations, and substrate feeding strategies were investigated on the biosynthesis of licoflavanone in recombinant E. coli. Consequently, the recombinant E. coli strain capable of improved dimethylallyl diphosphate (DMAPP) supply and suitable for prenylflavonoid biosynthesis increased licoflavanone titers to 142.1 mg/L in a shake flask and to 537.8 mg/L in a 1.3 L fermentor, which is the highest yield for any prenylflavonoids reported to date. These strategies proposed in this study provide a reference for initiating the production of high-value prenylflavonoids.

Telescoping a Prenyltransferase and a Diterpene Synthase to Transform Unnatural FPP Derivatives to Diterpenoids.

New diterpenoids are accessible from non-natural FPP derivatives as substrates for an enzymatic elongation cyclization cascade using the geranylgeranyl pyrophosphate synthase (GGPPS) from Streptomyces cyaneofuscatus and the spata-13,17-diene synthase (SpS) from Streptomyces xinghaiensis. This approach led to four new biotransformation products including three new cyclododecane cores and a macrocyclic ether. For the first time, a 1,12-terpene cyclization was observed when shifting the central olefinic double bond toward the geminial methyl groups creating a nonconjugated 1,4-diene.

Discovery of Prenyltransferase-Guided Hydroxyphenylacetic Acid Derivatives from Marine Fungus Penicillium sp. W21C371.

Traditional isolation methods often lead to the rediscovery of known natural products. In contrast, genome mining strategies are considered effective for the continual discovery of new natural products. In this study, we discovered a unique prenyltransferase (PT) through genome mining, capable of catalyzing the transfer of a prenyl group to an aromatic nucleus to form C-C or C-O bonds. A pair of new hydroxyphenylacetic acid derivative enantiomers with prenyl units, (±)-peniprenydiol A (1), along with 16 known compounds (2-17), were isolated from a marine fungus, Penicillium sp. W21C371. The separation of 1 using chiral HPLC led to the isolation of the enantiomers 1a and 1b. Their structures were established on the basis of extensive spectroscopic analysis, including 1D, 2D NMR and HRESIMS. The absolute configurations of the new compounds were determined by a modified Mosher method. A plausible biosynthetic pathway for 1 was deduced, facilitated by PT catalysis. In the in vitro assay, 2 and 3 showed promising inhibitory activity against Escherichia coli ß-glucuronidase (EcGUS), with IC50 values of 44.60 ± 0.84 µM and 21.60 ± 0.76 µM, respectively, compared to the positive control, D-saccharic acid 1,4-lactone hydrate (DSL). This study demonstrates the advantages of genome mining in the rational acquisition of new natural products.

Methods for the preparation and analysis of a bifunctional class II diterpene synthase, copalyl diphosphate synthase from Penicillium fellutanum.

Terpenes comprise the largest class of natural products and are used in applications spanning the areas of medicine, cosmetics, fuels, flavorings, and more. Copalyl diphosphate synthase from the Penicillium genus is the first bifunctional terpene synthase identified to have both prenyltransferase and class II cyclase activities within the same polypeptide chain. Prior studies of bifunctional terpene synthases reveal that these systems achieve greater catalytic efficiency by channeling geranylgeranyl diphosphate between the prenyltransferase and cyclase domains. A molecular-level understanding of substrate transit phenomena in these systems is highly desirable, but a long disordered polypeptide segment connecting the prenyltranferase and cyclase domains thwarts the crystallization of full-length enzymes. Accordingly, these systems are excellent candidates for structural analysis using cryo-electron microscopy (cryo-EM). Notably, these systems form hexameric or octameric oligomers, so the quaternary structure of the full-length enzyme may influence substrate transit between catalytic domains. Here, we describe methods for the preparation of bifunctional hexameric copalyl diphosphate synthase from Penicillium fellutanum (PfCPS). We also outline approaches for the preparation of cryo-EM grids, data collection, and data processing to yield two-dimensional and three-dimensional reconstructions.

Methods for the preparation and analysis of the diterpene cyclase fusicoccadiene synthase.

Prenyltransferases are terpene synthases that combine 5-carbon precursor molecules into linear isoprenoids of varying length that serve as substrates for terpene cyclases, enzymes that catalyze fascinating cyclization reactions to form diverse terpene natural products. Terpenes and their derivatives comprise the largest class of natural products and have myriad functions in nature and diverse commercial uses. An emerging class of bifunctional terpene synthases contains both prenyltransferase and cyclase domains connected by a disordered linker in a single polypeptide chain. Fusicoccadiene synthase from Phomopsis amygdali (PaFS) is one of the most well-characterized members of this subclass and serves as a model system for the exploration of structure-function relationships. PaFS has been structurally characterized using a variety of biophysical techniques. The enzyme oligomerizes to form a stable core of six or eight prenyltransferase domains that produce a 20-carbon linear isoprenoid, geranylgeranyl diphosphate (GGPP), which then transits to the cyclase domains for the generation of fusicoccadiene. Cyclase domains are in dynamic equilibrium between randomly splayed-out and prenyltransferase-associated positions; cluster channeling is implicated for GGPP transit from the prenyltransferase core to the cyclase domains. In this chapter, we outline the methods we are developing to interrogate the nature of cluster channeling in PaFS, including enzyme activity and product analysis assays, approaches for engineering the linker segment connecting the prenyltransferase and cyclase domains, and structural analysis by cryo-EM.

The putative prenyltransferase Nus1 is required for filamentation in the human fungal pathogen Candida albicans.

Candida albicans is a major fungal pathogen of humans that can cause serious systemic infections in vulnerable immunocompromised populations. One of its virulence attributes is its capacity to transition between yeast and filamentous morphologies, but our understanding of this process remains incomplete. Here, we analyzed data from a functional genomic screen performed with the C. albicans Gene Replacement And Conditional Expression collection to identify genes crucial for morphogenesis in host-relevant conditions. Through manual scoring of microscopy images coupled with analysis of each image using a deep learning-based method termed Candescence, we identified 307 genes important for filamentation in tissue culture medium at 37°C with 5% CO2. One such factor was orf19.5963, which is predicted to encode the prenyltransferase Nus1 based on sequence homology to Saccharomyces cerevisiae. We further showed that Nus1 and its predicted interacting partner Rer2 are important for filamentation in multiple liquid filament-inducing conditions as well as for wrinkly colony formation on solid agar. Finally, we highlight that Nus1 and Rer2 likely govern C. albicans morphogenesis due to their importance in intracellular trafficking, as well as maintaining lipid homeostasis. Overall, this work identifies Nus1 and Rer2 as important regulators of C. albicans filamentation and highlights the power of functional genomic screens in advancing our understanding of gene function in human fungal pathogens.

Maramycin, a Cytotoxic Isoquinolinequinone Terpenoid Produced through Heterologous Expression of a Bifunctional Indole Prenyltransferase/Tryptophan Indole-Lyase in S. albidoflavus.

Isoquinolinequinones represent an important family of natural alkaloids with profound biological activities. Heterologous expression of a rare bifunctional indole prenyltransferase/tryptophan indole-lyase enzyme from Streptomyces mirabilis P8-A2 in S. albidoflavus J1074 led to the activation of a putative isoquinolinequinone biosynthetic gene cluster and production of a novel isoquinolinequinone alkaloid, named maramycin (1). The structure of maramycin was determined by analysis of spectroscopic (1D/2D NMR) and MS spectrometric data. The prevalence of this bifunctional biosynthetic enzyme was explored and found to be a recent evolutionary event with only a few representatives in nature. Maramycin exhibited moderate cytotoxicity against human prostate cancer cell lines, LNCaP and C4-2B. The discovery of maramycin (1) enriched the chemical diversity of natural isoquinolinequinones and also provided new insights into crosstalk between the host biosynthetic genes and the heterologous biosynthetic genes in generating new chemical scaffolds.

Evaluating protein prenylation of human and viral CaaX sequences using a humanized yeast system.

Prenylated proteins are prevalent in eukaryotic biology (∼1-2% of proteins) and are associated with human disease, including cancer, premature aging and infections. Prenylated proteins with a C-terminal CaaX sequence are targeted by CaaX-type prenyltransferases and proteases. To aid investigations of these enzymes and their targets, we developed Saccharomyces cerevisiae strains that express these human enzymes instead of their yeast counterparts. These strains were developed in part to explore human prenyltransferase specificity because of findings that yeast FTase has expanded specificity for sequences deviating from the CaaX consensus (i.e. atypical sequence and length). The humanized yeast strains displayed robust prenyltransferase activity against CaaX sequences derived from human and pathogen proteins containing typical and atypical CaaX sequences. The system also recapitulated prenylation of heterologously expressed human proteins (i.e. HRas and DNAJA2). These results reveal that substrate specificity is conserved for yeast and human farnesyltransferases but is less conserved for type I geranylgeranyltransferases. These yeast systems can be easily adapted for investigating the prenylomes of other organisms and are valuable new tools for helping define the human prenylome, which includes physiologically important proteins for which the CaaX modification status is unknown.

Increasing structure diversity of farnesylated chalcones by a fungal aromatic prenyltransferase.

Farnesylated chalcones were favored by researchers due to their different biological activities. However, only five naturally occurring farnesylated chalcones were described in the literature until now. Here, the farnesylation of six chalcones by the Aspergillus terreus aromatic prenyltransferase AtaPT was reported. Fourteen monofarnesylated chalcones (1F1-1F5, 2F1-2F3, 3F1, 3F2, 4F1, 4F2, 5F1, 6F1, and 6F2) and a difarnesylated product (2F3) were obtained, enriching the diversity of natural farnesylated chalcones significantly. Ten of them are C-farnesylated products, which complement O-farnesylated chalcones by chemical synthesis. Fourteen products have not been reported prior to this study. Nine of the produced compounds (1F2-1F5, 2F1-2F3, 5F1, and 6F1) exhibited inhibitory effect on α-glucosidase with IC50 values ranging from 24.08 ± 1.44 to 190.0 ± 0.28 µM. Among them, compounds 2F3 with IC50 value at 24.08 ± 1.44 µM and 1F4 with IC50 value at 30.09 ± 0.59 µM showed about 20 times stronger than the positive control acarbose with an IC50 at 536.87 ± 24.25 µM in α-glucosidase inhibitory assays.

Genome Mining Reveals a UbiA-Type Prenyltransferase Access to Farnesylation of Diketopiperazines.

UbiA-type prenyltransferases (PTases) are significant enzymes that lead to structurally diverse meroterpenoids. Herein, we report the identification and characterization of an undescribed UbiA-type PTase, FtaB, that is responsible for the farnesylation of indole-containing diketopiperazines (DKPs) through genome mining. Heterologous expression of the fta gene cluster and non-native pathways result in the production of a series of new C2-farnesylated DKPs. This study broadens the reaction scope of UbiA-type PTases and expands the chemical diversity of meroterpenoids.

Water-deficit stress induces prenylated stilbenoid production and affects biomass in peanut hairy roots: Exploring the role of stilbenoid prenyltransferase downregulation.

The peanut plant is one of the most economically important crops around the world. Abiotic stress, such as drought, causes over five hundred million dollars in losses in peanut production per year. Peanuts are known to produce prenylated stilbenoids to counteract biotic stress. However, their role in abiotic stress tolerance has not been elucidated. To address this issue, hairy roots with the capacity to produce prenylated stilbenoids were established. An RNA-interference (RNAi) molecular construct targeting the stilbenoid-specific prenyltransferase AhR4DT-1 was designed and expressed via Agrobacterium rhizogenes-mediated transformation in hairy roots of peanut cultivar Georgia Green. Two transgenic hairy roots with the RNAi molecular construct were established, and the downregulation of AhR4DT-1 was validated using reverse transcriptase quantitative PCR. To determine the efficacy of the RNAi-approach in modifying the levels of prenylated stilbenoids, the hairy roots were co-treated with methyl jasmonate, hydrogen peroxide, cyclodextrin, and magnesium chloride to induce the production of stilbenoids and then the stilbenoids were analyzed in extracts of the culture medium. Highly reduced levels of prenylated stilbenoids were observed in the RNAi hairy roots. Furthermore, the hairy roots were evaluated in a polyethylene glycol (PEG) assay to assess the role of prenylated stilbenoids on water-deficit stress. Upon PEG treatment, stilbenoids were induced and secreted into the culture medium of RNAi and wild-type hairy roots. Additionally, the biomass of the RNAi hairy roots decreased by a higher amount as compared to the wild-type hairy roots suggesting that prenylated stilbenoids might play a role against water-deficit stress.

Fungal Prenyltransferase AnaPT and Its F265 Mutants Catalyze the Dimethylallylation at the Nonaromatic Carbon of Phloretin.

Phloretin is widely found in fruit and shows various biological activities. Here, we demonstrate the dimethylallylation, geranylation, and farnesylation, particularly the first dimethylallylation at the nonaromatic carbon of phloretin (1) by the fungal prenyltransferase AnaPT and its mutants. F265 was identified as a key amino acid residue related to dimethylallylation at the nonaromatic carbon of phloretin. Mutants AnaPT_F265D, AnaPT_F265G, AnaPT_F265P, AnaPT_F265C, and AnaPT_F265Y were discovered to generally increase prenylation activity toward 1. AnaPT_F265G catalyzes the O-geranylation selectively at the C-2' hydroxyl group, which involves an intramolecular hydrogen bond with the carbonyl group of 1. Seven products, 1D5, 1D7-1D9, 1G2, 1G4, and 1F2, have not been reported prior to this study. Twelve compounds, 1D3-1D9, 1G1-1G3, and 1F1-1F2, exhibited potential inhibitory effects on α-glucosidase with IC50 values ranging from 11.45 ± 0.87 to 193.80 ± 6.52 µg/mL. Among them, 1G1 with an IC50 value of 11.45 ± 0.87 µg/mL was the most potential α-glucosidase inhibitor, which is about 30 times stronger than the positive control acarbose with an IC50 value of 346.63 ± 15.65 µg/mL.

A prenyltransferase participates in the biosynthesis of anthraquinones in Rubia cordifolia.

Anthraquinones (AQs) constitute the largest group of natural quinones, which are used as safe natural dyes and have many pharmaceutical applications. In plants, AQs are biosynthesized through two main routes: the polyketide pathway and the shikimate pathway. The latter primarily forms alizarin-type AQs, and the prenylation of 1,4-dihydroxy-2-naphthoic acid (DHNA) is the first pathway-specific step. However, the prenyltransferase (PT) responsible for this key step remains uncharacterized. In this study, the cell suspension culture of Madder (Rubia cordifolia), a plant rich in alizarin-type AQs, was discovered to be capable of prenylating DHNA to form 2-carboxyl-3-prenyl-1,4-naphthoquinone and 3-prenyl-1,4-naphthoquinone. Then, a candidate gene belonging to the UbiA superfamily, R. cordifoliadimethylallyltransferase 1 (RcDT1), was shown to account for the prenylation activity. Substrate specificity studies revealed that the recombinant RcDT1 recognized naphthoic acids primarily, followed by 4-hydroxyl benzoic acids. The prenylation activity was strongly inhibited by 1,2- and 1,4-dihydroxynaphthalene. RcDT1 RNA interference significantly reduced the AQs content in R. cordifolia callus cultures, demonstrating that RcDT1 is required for alizarin-type AQs biosynthesis. The plastid localization and root-specific expression further confirmed the participation of RcDT1 in anthraquinone biosynthesis. The phylogenetic analyses of RcDT1 and functional validation of its rubiaceous homologs indicated that DHNA-prenylation activity evolved convergently in Rubiaceae via recruitment from the ubiquinone biosynthetic pathway. Our results demonstrate that RcDT1 catalyzes the first pathway-specific step of alizarin-type AQs biosynthesis in R. cordifolia. These findings will have profound implications for understanding the biosynthetic process of the anthraquinone ring derived from the shikimate pathway.

Biochemical characterization of a multiple prenyltransferase from Tolypocladium inflatum.

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.

Enzymatic properties and immobilization of a thermostable prenyltransferase from Aspergillus fumigatiaffinis for the production of prenylated naringenin.

Prenyltransferases catalyze the synthesis of prenylated flavonoids, providing these with greater lipid solubility, biological activity, and availability. In this study, a thermostable prenyltransferase (AfPT) from Aspergillus fumigatiaffinis was cloned and expressed in Escherichia coli. By optimizing induction conditions, the expression level of AfPT reached 39.3 mU/mL, which was approximately 200 % of that before optimization. Additionally, we determined the enzymatic properties of AfPT. Subsequently, AfPT was immobilized on carboxymethyl cellulose magnetic nanoparticles (CMN) at a maximum load of 0.6 mg/mg. Optimal activity of CMN-AfPT was achieved at pH 8.0 and 55 °C. Thermostability assays showed that the residual activity of CMN-AfPT was greater than 50 % after incubation at 55 °C for 4 h. Km and Vmax of CMN-AfPT for naringenin were 0.082 mM and 5.57 nmol/min/mg, respectively. The Kcat/Km ratio of CMN-AfPT was higher than that of AfPT. Residual prenyltransferase activity of CMN-AfPT remained higher than 70 % even after 30 days of storage. Further, CMN-AfPT retained 68 % of its original activity after 10 cycles of reuse. Compared with free AfPT, CMN-AfPT showed higher catalytic efficiency, thermostability, metal ion tolerance, substrate affinity, storage stability, and reusability. Our study presents a thermostable prenyltransferase and its immobilized form for the production of prenylated flavonoids in vitro.

Substrate-Dependent Alteration in the C- and O-Prenylation Specificities of Cannabis Prenyltransferase.

CsPT4 is an aromatic prenyltransferase that synthesizes cannabigerolic acid (CBGA), the key intermediate of cannabinoid biosynthesis in Cannabis sativa, from olivetolic acid (OA) and geranyl diphosphate (GPP). CsPT4 has a catalytic potential to produce a variety of CBGA analogs via regioselective C-prenylation of aromatic substrates having resorcylic acid skeletons including bibenzyl 2,4-dihydroxy-6-phenylethylbenzoic acid (DPA). In this study, we further investigated the substrate specificity of CsPT4 using phlorocaprophenone (PCP) and 2',4',6'-trihydroxydihydrochalcone (THDC), the isomers of OA and DPA, respectively, and demonstrated that CsPT4 catalyzed both C-prenylation and O-prenylation reactions on PCP and THDC that share acylphloroglucinol substructures. Interestingly, the kinetic parameters of CsPT4 for these substrates differed depending on whether they underwent C-prenylation or O-prenylation, suggesting that this enzyme utilized different substrate-binding modes suitable for the respective reactions. Aromatic prenyltransferases that catalyze O-prenylation are rare in the plant kingdom, and CsPT4 was notable for altering the reaction specificity between C- and O-prenylations depending on the skeletons of aromatic substrates. We also demonstrated that enzymatically synthesized geranylated acylphloroglucinols had potent antiausterity activity against PANC-1 human pancreatic cancer cells, with 4'-O-geranyl THDC being the most effective. We suggest that CsPT4 is a valuable catalyst to generate biologically active C- and O-prenylated molecules that could be anticancer lead compounds.

Vitamin K converting enzyme UBIAD1 plays an important role in osteogenesis and chondrogenesis in mice.

Dietary vitamin K1 (phylloquinone: PK) and menaquinone (MK-n) are converted to menadione (MD) in the small intestine and then translocated to various tissues where they are converted to vitamin K2 (menaquinone-4: MK-4) by UbiA prenyltransferase domain containing protein 1 (UBIAD1). MK-4 is effective in bone formation and is used to treat osteoporosis in Japan. UBIAD1 is expressed in bone and osteoblasts and shows conversion to MK-4, but the role of UBIAD1 in osteogenesis is unknown. In this study, we investigated the function of UBIAD1 in osteogenesis using a tamoxifen-dependent UBIAD1-deficient mouse model. When UBIAD1 deficiency was induced from the first week of life, the femur was significantly shortened, and bone mineral density (BMD) was reduced. In addition, the expression of bone and chondrocyte matrix proteins and chondrocyte differentiation factors was significantly decreased. In primary cultured chondrocytes, chondrocyte differentiation was significantly reduced by UBIAD1 deficiency. These results suggest that UBIAD1 is an important factor for the regulation of chondrocyte proliferation and differentiation during osteogenesis.