ABSTRACT
Polyethylene terephthalate (PET), one of the most widely used plastics in the world, causes serious environmental problems. Recently, scientists have been focused on the enzymatic degradation of PET, an environmentally friendly method that offers an attractive approach to the degradation and recycling of PET. In this work, PET hydrolase from Streptomyces sp. W2061 was biochemically characterized, and the biodegradation of PET was performed using the PET model substrate bis (2- hydroxyethyl terephthalate) (BHET). PET hydrolase has an isoelectric point of 5.84, and a molecular mass of about 50.31 kDa. The optimum pH and temperature were 7.0 and 40°C, respectively. LC-MS analysis of the enzymatic products showed that the PET hydrolase successfully degraded a single ester bond of BHET, leading to the formation of MHET. Furthermore, in silico characterization of the PET hydrolase protein sequence and its predicted three-dimensional structure was designed and compared with the well-characterized IsPETase from Ideonella sakaiensis. The structural analysis showed that the (Gly-x1-Ser-x2-Gly) serine hydrolase motif and the catalytic triad (Ser, Asp, and His) were conserved in all sequences. In addition, we integrated molecular dynamics (MD) simulations to analyze the variation in the structural stability of the PET hydrolase in the absence and presence of BHET. These simulations showed the formation of a stable complex between the PET hydrolase and BHET. To the best of our knowledge, this is the first study on Streptomyces sp. W2061 to investigate the BHET degradation activity of PET hydrolase, which has potential application in the biodegradation of plastics in the environment.
ABSTRACT
Although previous studies have examined the signaling pathway involved in melanogenesis through which ultraviolet (UV) or α-melanocyte-stimulating hormones (α-MSH) stimuli act as key inducers to produce melanin at the stratum basal layer of the epidermis, the signaling pathway regulating melanogenesis is still controversial. This study reports that α-MSH, not UVA and UVB, acted as a major stimulus of melanogenesis in B16F10 melanoma cells. Signaling pathway analysis using gene knockdown technology and chemical inhibitors, the mitogen-activated protein kinase kinase (MEK)/extracellular signal-regulated kinase (ERK)/p90 ribosomal S6 kinase 2 (RSK2) played an important role in melanogenesis. Unexpectedly, LY294002, a PI3K inhibitor, increased melanogenesis without UV or α-MSH stimulation, suggesting that the PI3K/AKT signaling pathway may not be a major signaling pathway for melanogenesis. Chemical inhibition of the MEKs/ERKs/RSK2 signaling pathway using U0126 or BI-D1870 suppressed melanogenesis by stimulation of UVA or α-MSH stimulation, or both. In particular, the genetic depletion of RSK2 or constitutive active (CA)-RSK2 overexpression showed that RSK2 plays a key role in melanogenesis. Interestingly, forkhead box protein O4 (FOXO4) was phosphorylated by RSK2, resulting in the increase of FOXO4's transactivation activity. Notably, the FOXO4 mutant harboring serine-to-alanine replacement at the phosphorylation sites totally abrogated the transactivation activity and reduced melanin production, indicating that RSK2-mediated FOXO4 activity plays a key role in melanogenesis. Furthermore, kaempferol, a flavonoid inhibiting the RSK2 activity, suppressed melanogenesis. In addition, FOXO4-wt overexpression showed that FOXO4 enhance melanin synthesis. Overall, the RSK2-FOXO4 signaling pathway plays a key role in modulating melanogenesis.
Subject(s)
Melanins , Pteridines , Ribosomal Protein S6 Kinases, 90-kDa , Signal Transduction , alpha-MSH , Ribosomal Protein S6 Kinases, 90-kDa/metabolism , Ribosomal Protein S6 Kinases, 90-kDa/genetics , Melanins/biosynthesis , Melanins/metabolism , Animals , alpha-MSH/metabolism , alpha-MSH/pharmacology , Mice , Cell Line, Tumor , Forkhead Transcription Factors/metabolism , Forkhead Transcription Factors/genetics , Ultraviolet Rays , Morpholines/pharmacology , Chromones/pharmacology , Nitriles/pharmacology , Butadienes/pharmacology , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Melanoma, Experimental/metabolism , MelanogenesisABSTRACT
BACKGROUND: Excessive stress, a major problem in modern societies, affects people of all ages worldwide. Corticosterone is one of the most abundant hormones secreted during stressful conditions and is associated with various dysfunctions in the body. In particular, we aimed to investigate the protective effects of hygrolansamycin C (HYGC) against corticosterone-induced cellular stress, a manifestation of excessive stress prevalent in contemporary societies. METHODS: We isolated HYGC from Streptomyces sp. KCB17JA11 and subjected PC12 cells to corticosterone-induced stress. The effects of HYGC were assessed by measuring autophagy and the expression of mitogen-activated protein kinase (MAPK) phosphorylation-related genes. We used established cellular and molecular techniques to analyze protein levels and pathways. RESULTS: HYGC effectively protected cells against corticosterone-induced injury. Specifically, it significantly reduced corticosterone-induced oxidative stress and inhibited the expression of autophagy-related proteins induced by corticosterone, which provided mechanistic insight into the protective effects of HYGC. At the signaling level, HYGC suppressed c-Jun N-terminal kinase and extracellular signal-regulated kinase phosphorylation and p38 activation. CONCLUSIONS: HYGC is a promising candidate to counteract corticosterone-induced apoptosis and oxidative stress. Autophagy and MAPK pathway inhibition contribute to the protective effects of HYGC. Our findings highlight the potential of HYGC as a therapeutic agent for stress-related disorders and serve as a stepping stone for further exploration and development of stress management strategies.
Subject(s)
Corticosterone , p38 Mitogen-Activated Protein Kinases , Rats , Animals , Humans , Corticosterone/toxicity , p38 Mitogen-Activated Protein Kinases/metabolism , MAP Kinase Signaling System , Oxidative Stress , Signal Transduction , Extracellular Signal-Regulated MAP Kinases/metabolism , Apoptosis , AutophagyABSTRACT
Cancer cells often exhibit resistance to apoptotic cell death, but they may be vulnerable to other types of cell death. Elucidating additional mechanisms that govern cancer cell death is crucial for developing new therapies. Our research identified cyclic AMP-responsive element-binding protein 3 (CREB3) as a crucial regulator and initiator of a unique cell death mechanism known as karyoptosis. This process is characterized by nuclear shrinkage, deformation, and the loss of nuclear components following nuclear membrane rupture. We found that the N-terminal domain (aa 1-230) of full-length CREB3 (CREB3-FL), which is anchored to the nuclear inner membrane (INM), interacts with lamins and chromatin DNA. This interaction maintains a balance between the outward force exerted by tightly packed DNA and the inward constraining force, thereby preserving INM integrity. Under endoplasmic reticulum (ER) stress, aberrant cleavage of CREB3-FL at the INM leads to abnormal accumulation of the cleaved form of CREB3 (CREB3-CF). This accumulation disrupts the attachment of CREB3-FL to the INM, resulting in sudden rupture of the nuclear membrane and the onset of karyoptosis. Proteomic studies revealed that CREB3-CF overexpression induces a DNA damage response akin to that caused by UVB irradiation, which is associated with cellular senescence in cancer cells. These findings demonstrated that the dysregulation of CREB3-FL cleavage is a key factor in karyoptotic cell death. Consequently, these findings suggest new therapeutic strategies in cancer treatment that exploit the process of karyoptosis.
Subject(s)
Cyclic AMP Response Element-Binding Protein , Nuclear Envelope , Proteomics , Apoptosis , DNA , Nuclear Envelope/metabolism , Humans , Cell Line, Tumor , Cyclic AMP Response Element-Binding Protein/metabolismABSTRACT
The pluramycin family of natural products has diverse substituents at the C2 position, which are closely related to their biological activity. Therefore, it is important to understand the biosynthesis of C2 substituents. In this study, we describe the biosynthesis of C2 moieties in Streptomyces sp. W2061, which produces kidamycin and rubiflavinone C-1, containing anthrapyran aglycones. Sequence analysis of the loading module (Kid13) of the PKS responsible for the synthesis of these anthrapyran aglycones is useful for confirming the incorporation of atypical primer units into the corresponding products. Kid13 is a ketosynthase-like decarboxylase (KSQ)-type loading module with unusual dual acyltransferase (AT) domains (AT1-1 and AT1-2). The AT1-2 domain primarily loads ethylmalonyl-CoA and malonyl-CoA for rubiflavinone and kidamycinone and rubiflavinone, respectively; however, the AT1-1 domain contributed to the functioning of the AT1-2 domain to efficiently load ethylmalonyl-CoA for rubiflavinone. We found that the dual AT system was involved in the production of kidamycinone, an aglycone of kidamycin, and rubiflavinone C-1 by other shared biosynthetic genes in Streptomyces sp. W2061. This study broadens our understanding of the incorporation of atypical primer units into polyketide products.
ABSTRACT
Liquid chromatography-mass spectrometry (LC-MS/MS)-based molecular networking analysis was applied to Streptomyces sp. MC16. The automatic classification of the MolNetEnhancer module revealed that its major constituent was an angucycline derivative. By targeted isolation of unique clusters in the molecular network, which showed different patterns from typical angucycline compounds, two new N-acetylcysteine-attached angucycline derivatives (1 and 2) were isolated. The structures were elucidated based on intensive NMR analysis and high-resolution electrospray ionization mass spectrometry (HR-ESI-MS). All isolated compounds (1-4) were tested for their inhibitory effects on the proliferation of A431, A549, and HeLa cell lines. Antibiotics 100-1 (3) and vineomycinone B2 (4) showed moderate inhibitory effects on these three cell lines with IC50 values ranging from 18.5 to 59.0 µM, while compounds 1 and 2 with an additional N-acetylcysteine residue showed weak inhibitory effects only on the HeLa cell line with IC50 values of 54.7 and 65.2 µM, respectively.
ABSTRACT
A recently bioinformatic analysis of genomic sequences of fungi indicated that fungi are able to produce more secondary metabolites than expected. Despite their potency, many biosynthetic pathways are silent in the absence of specific culture conditions or chemical cues. To access cryptic metabolism, 108 fungal strains isolated from various sites were cultured with or without Streptomyces sp. 13F051 which mainly produces trichostatin analogues, followed by comparison of metabolic profiles using LC-MS. Among the 108 fungal strains, 14 produced secondary metabolites that were not recognized or were scarcely produced in mono-cultivation. Of these two fungal strains, Myrmecridium schulzeri 15F098 and Scleroconidioma sphagnicola 15S058 produced four new compounds (1-4) along with a known compound (5), demonstrating that all four compounds were produced by physical interaction with Streptomyces sp. 13F051. Bioactivity evaluation indicated that compounds 3-5 impede migration of MDA-MB-231 breast cancer cells.
Subject(s)
Actinobacteria , Histone Deacetylase Inhibitors , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylase Inhibitors/metabolism , Coculture Techniques , Actinobacteria/genetics , Actinobacteria/metabolism , Fungi/metabolism , Metabolome , Secondary Metabolism/geneticsABSTRACT
The pluramycin family of antibiotics comprises angucycline compounds derived from actinomycetes that possess anticancer and antibacterial properties. Pluramycins are structurally characterized by two aminoglycosides linked by a carbon-carbon bond next to the γ-pyrone angucycline backbone. Kidamycins (3, 4) and rubiflavins (6-9) were screened through liquid chromatography-mass spectrometry analysis of the crude extracts of Streptomyces sp. W2061, which was cultured in complex media under phosphate-limiting conditions. Newly isolated rubiflavin G (7) and photoactivated compounds (8, 9) were characterized using exhaustive 1D and 2D nuclear magnetic resonance analysis. The cytotoxicity of kidamycin (3), photokidamycin (4), and photorubiflavin G (8) was determined using two human breast cancer cell lines-MCF7 and MDA-MB-231. Compared to MCF7 cells, MDA-MB-231 cells were more sensitive to the active compounds, and photokidamycin (4) considerably inhibited MCF7 and MDA-MB-231 cell growth (IC50 = 3.51 and 0.66 µM, respectively).
Subject(s)
Antineoplastic Agents , Breast Neoplasms , Streptomyces , Humans , Female , Streptomyces/chemistry , Breast Neoplasms/drug therapy , Aminoglycosides , Anti-Bacterial Agents/pharmacology , Anti-Bacterial Agents/chemistry , Carbon , Cell Line, Tumor , Antineoplastic Agents/pharmacology , Antineoplastic Agents/chemistryABSTRACT
Agar is a galactan and a major component of the red algal cell wall. Agar is metabolized only by specific microorganisms. The final step of the ß-agarolytic pathway is mediated by α-neoagarooligosaccharide hydrolase (α-NAOSH), which cleaves neoagarobiose to D-galactose and 3,6-anhydro-α-L-galactose. In the present study, two α-NAOSHs, SCO3481 and SCO3479, were identified in Streptomyces coelicolor A3(2). SCO3481 (370 amino acids, 41.12 kDa) and SCO3479 (995 amino acids, 108.8 kDa) catalyzed the hydrolysis of the α-(1,3) glycosidic bonds of neoagarobiose, neoagarotetraose, and neoagarohexaose at the nonreducing ends, releasing 3,6-anhydro-α-L-galactose. Both were intracellular proteins without any signal peptides for secretion. Similar to all α-NAOSHs reported to date, SCO3481 belonged to the glycosyl hydrolase (GH) 117 family and formed dimers. On the other hand, SCO3479 was a large monomeric α-NAOSH belonging to the GH2 family with a ß-galactosidase domain. SCO3479 also clearly showed ß-galactosidase activity toward lactose and artificial substrates, but SCO3481 did not. The optimum conditions for α-NAOSH were pH 6.0 and 25 °C for SCO3481, and pH 6.0 and 30 °C for SCO3479. Enzymatic activity was enhanced by Co2+ for SCO3481 and Mg2+ for SCO3479. The ß-galactosidase activity of SCO3479 was maximum at pH 7.0 and 50 °C and was increased by Mg2+. Many differences were evident in the kinetic parameters of each enzyme. Although SCO3481 is typical of the GH117 family, SCO3479 is a novel α-NAOSH that was first reported in the GH2 family. SCO3479, a unique bifunctional enzyme with α-NAOSH and ß-galactosidase activities, has many advantages for industrial applications. KEY POINTS: ⢠SCO3481 is a dimeric α-neoagarooligosaccharide hydrolase belonging to GH117. ⢠SCO3479 is a monomeric α-neoagarooligosaccharide hydrolase belonging to GH2. ⢠SCO3479 is a novel and unique bifunctional enzyme that also acts as a ß-galactosidase.
Subject(s)
Streptomyces coelicolor , Streptomyces coelicolor/genetics , Streptomyces coelicolor/metabolism , Galactose/chemistry , Agar/metabolism , Glycoside Hydrolases/metabolism , Galactosidases/metabolism , beta-GalactosidaseABSTRACT
A new secondary metabolite, ulleungdolin (1), was isolated from the co-culture of an actinomycete, Streptomyces sp. 13F051, and a fungus, Leohumicola minima 15S071. Based on the NMR, UV, and MS data, it was deduced that the planar structure of 1 comprised an isoindolinone (IsoID) with an octanoic acid, a tripeptide, and a sugar. The tripeptide has the unprecedented amino acids norcoronamic acid, 3-hydroxy-glutamine, and 4-hydroxy-phenylglycine and is linked by a C-N bond with IsoID. The absolute configurations were determined by chemical derivatization, extensive spectroscopic methods, and electronic circular dichroism calculations and supported by bioinformatic analyses. Bioactivity evaluation studies indicated that 1 had an antimigration effect on MDA-MB-231 breast cancer cells.
Subject(s)
Ascomycota , Polyketides , Streptomyces , Streptomyces/chemistry , Polyketides/pharmacology , Polyketides/chemistry , Coculture Techniques , Molecular Structure , PeptidesABSTRACT
Six ansamycin derivatives were isolated from the culture broth of Streptomyces sp. KCB17JA11, including four new hygrolansamycins A-D (1-4) and known congeners divergolide O (5) and hygrocin C (6). Compounds 1-5 featured an unusual six-membered O-heterocyclic moiety. The isolation workflow was guided by a Molecular Networking-based dereplication strategy. The structures of 1-4 were elucidated using NMR and HRESIMS experiments, and the absolute configuration was established by the Mosher's method. Compound 2 exhibited mild cytotoxicity against five cancer cell lines with IC50 values ranging from 24.60 ± 3.37 µM to 49.93 ± 4.52 µM.
Subject(s)
Streptomyces , Streptomyces/chemistry , Macrolides/chemistry , Molecular Structure , Anti-Bacterial Agents/pharmacology , Lactams, MacrocyclicABSTRACT
Kidamycins belong to the pluramycin family of antitumor antibiotics that contain di-C-glycosylated angucycline. Owing to its interesting biological activity, several synthetic derivatives of kidamycins are currently being developed. However, the synthesis of these complex structural compounds with unusual C-glycosylated residues is difficult. In the kidamycin-producing Streptomyces sp. W2061 strain, the genes encoding the biosynthetic enzymes responsible for the structural features of kidamycin were identified. Two glycosyltransferase-coding genes, kid7 and kid21, were found in the kidamycin biosynthetic gene cluster (BGC). Gene inactivation studies revealed that the subsequent glycosylation steps occurred in a sequential manner, in which Kid7 first attached N,N-dimethylvancosamine to the C10 position of angucycline aglycone, following which Kid21 transferred an anglosamine moiety to C8 of the C10-glycosylated angucycline. Therefore, this is the first report to reveal the sequential biosynthetic steps of the unique C-glycosylated amino-deoxyhexoses of kidamycin. Additionally, we confirmed that all three methyltransferases (Kid4, Kid9, and Kid24) present in this BGC were involved in the biosynthesis of these amino-deoxyhexoses, N,N-dimethylvancosamine and anglosamine. Aglycone compounds and the mono-C-glycosylated compound obtained in this process will be used as substrates for the development of synthetic derivatives in the future.
ABSTRACT
Two new polyketide glycosides jejuketomycins A (1) and B (2), were isolated from a culture of Streptomyces sp. KCB15JA151. Their chemical structures including the absolute configurations were determined by detailed analyses of the NMR and HRMS data and ECD calculations and spectral data. Compounds 1 and 2 possess an unusual 6/6/8 tricyclic ring system. Biological evaluation with the wound healing assay and time-lapse cell tracking analysis revealed that compounds 1 and 2 have significant inhibitory activities against cancer cell migration with low cytotoxicity.
ABSTRACT
Zingerone (vanillylacetone; 4-hydroxy-3-methoxyphenylethyl methyl ketone) is a key component responsible for the pungency of ginger (Zingiber officinale). In this study, it was confirmed that a type III polyketide synthase (PKS) gene (pmpks) from Piper methysticum exhibits feruloyl-CoA-preferred benzalacetone synthase (BAS) activity. Based on these results, we constructed an artificial biosynthetic pathway for zingerone production from supplemented ferulic acid with 4-coumarate CoA ligase (4CL), PmPKS, and benzalacetone reductase (BAR). Furthermore, a de novo pathway for the production of zingerone was assembled using six heterologous genes, encoding tyrosine ammonia-lyase (optal), cinnamate-4-hydroxlase (sam5), caffeic acid O-methyltransferase (com), 4CL (4cl2nt), BAS (pmpks), and BAR (rzs1), in Escherichia coli. Using the engineered l-tyrosine-overproducing E. coli ΔCOS4 strain as a host, a maximum yield of 24.03 ± 2.53 mg/L zingerone was achieved by complete de novo synthesis.
Subject(s)
Biosynthetic Pathways , Kava , Butanones , Escherichia coli/genetics , Guaiacol/analogs & derivativesABSTRACT
Three new trichostatin analogues, ulleunganilines A-C (1-3), and seven known trichostatins (4-10) were isolated from cultures of Streptomyces sp. 13F051. NMR, UV, and MS data indicated that the planar structures of 1-3 consisted of modified side chains in the trichostatic acid moiety. The absolute configuration of the 2,4-dimethyl-branched carbon chains in 1 and 2 was determined by the PGME method, while the amino acid group in 3 was identified by advanced Marfey's method. Based on the structure of the modified side chains, the origin of 1-3 is proposed. Further experiments indicated that 1 and 3 displayed moderate histone deacetylase inhibitory activity, suggesting that not only the hydroxamate group but also the N,N-dimethyl group were essential for the inhibitory activity.
Subject(s)
Histone Deacetylase Inhibitors/pharmacology , Hydroxamic Acids/pharmacology , Cell Line, Tumor , Histone Deacetylase Inhibitors/isolation & purification , Humans , Hydroxamic Acids/isolation & purification , Molecular Structure , Republic of Korea , Soil Microbiology , Streptomyces/chemistryABSTRACT
A cDNA clone (named pnpks), which shows high homology to the known chalcone synthase (CHS)-like type III PKS, was obtained from the leaves of Piper nigrum. The PnPKS protein with ferulic acid catalyzed lactonization instead of chalcone or stilbene formation. The new product was characterized as a styrylpyrone, 11-methoxy-bisnoryangonin, which is the lactonization compound of a linear triketide formed as the reaction product of PnPKS protein with ferulic acid. These results show that pnpks encodes a styrylpyrone synthase (SPS)-like PKS that catalyzes two-chain elongation with feruloyl CoA-linked starter substrates. Although these styrylpyrone compounds are promising for use in human healthcare, they are mainly obtained by extraction from raw plant or mushroom sources. For de novo synthesis of 11-methoxy-bisnoryangonin in the heterologous host Escherichia coli from a simple sugar as a starter, the artificial biosynthetic pathway contained five genes: optal, sam5, com, and 4cl2nt, along with the pnpks gene. The engineered L-tyrosine overproducing E. coli ∆COS1 strain, in which five biosynthetic genes were cloned into two vectors, pET-opT5M and pET22-4P, was cultured for 24 h in a minimal glucose medium containing ampicillin and kanamycin. As a result, 11-methoxy-bisnoryangonin production of up to 52.8 mg/L was achieved, which is approximately 8.5-fold higher than that in the parental E. coli strain harboring a plasmid for 11-methoxy-bisnoryangonin biosynthesis. As a potential styrylpyrone compound, 11-methoxy-bisnoryangonin, was successfully produced in E. coli from a simple glucose medium, and its production titer was also increased using engineered strains. This study provides a useful reference for establishing the biological manufacture of styrylpyrone compounds.
ABSTRACT
Two angucyclines, pseudonocardones D (1) and E (2), were isolated from Streptomyces sp. KCB15JA151. The planar structure was elucidated by comprehensive spectroscopic analysis. The absolute configuration of the sugar unit was determined based on the basis of coupling constants, ROESY, chemical derivatization and HPLC analysis. The biological activities of compounds 1 and 2 were examined by performing a computational target prediction, which led to tests of the antiestrogenic activity. The result suggested that compound 1 might be an ERα antagonist.
Subject(s)
Estrogen Receptor alpha/antagonists & inhibitors , Glucuronic Acid/pharmacology , Streptomyces/chemistry , Dose-Response Relationship, Drug , Estrogen Receptor alpha/metabolism , Glucuronic Acid/chemistry , Glucuronic Acid/isolation & purification , Humans , Molecular Structure , Structure-Activity RelationshipABSTRACT
Actinobacteria utilize various polysaccharides in the soil as carbon source by degrading them via extracellular hydrolytic enzymes. Agarose, a marine algal polysaccharide composed of D-galactose and 3,6-anhydro-L-galactose (AHG), is one of the carbon sources used by S. coelicolor A3(2). However, little is known about agar hydrolysis in S. coelicolor A3(2), except that the regulation of agar hydrolysis metabolism is strongly inhibited by glucose as in the catabolic pathways of other polysaccharides. In this study, we elucidated the role of DagR in regulating the expression of three agarase genes (dagA, dagB, and dagC) in S. coelicolor A3(2) by developing a dagR-deletion mutant (Δsco3485). We observed that the Δsco3485 mutant had increased mRNA level of the agarolytic pathway genes and 1.3-folds higher agarase production than the wild type strain, indicating that the dagR gene encodes a cluster-suited repressor. Electrophoretic mobility shift assay revealed that DagR bound to the upstream regions of the three agarase genes. DNase 1 footprinting analysis demonstrated that a palindromic sequence present in the upstream region of the three agarase genes was essential for DagR-binding. Uniquely, the DNA-binding activity of DagR was inhibited by AHG, one of the final degradation products of agarose. AHG-induced agarase production was not observed in the Δsco3485 mutant, as opposed to that in the wild type strain. Therefore, DagR acts as a repressor that binds to the promoter region of the agarase genes, inhibits gene expression at the transcriptional level, and is derepressed by AHG. This is the first report on the regulation of gene expression regarding agar metabolism in S. coelicolor A3(2).
ABSTRACT
Bacterial cytochrome P450 (CYP) enzymes are responsible for the hydroxylation of diverse endogenous substances with a heme molecule used as a cofactor. This study characterized two CYP154C3 proteins from Streptomyces sp. W2061 (CYP154C3-1) and Streptomyces sp. KCCM40643 (CYP154C3-2). The enzymatic activity assays of both CYPs conducted using heterologous redox partners' putidaredoxin and putidaredoxin reductase showed substrate flexibility with different steroids and exhibited interesting product formation patterns. The enzymatic characterization revealed good activity over a pH range of 7.0 to 7.8 and the optimal temperature range for activity was 30 to 37°C. The major product was the C16-hydroxylated product and the kinetic profiles and patterns of the generated hydroxylated products differed between the two enzymes. Both enzymes showed a higher affinity toward progesterone, with CYP154C3-1 demonstrating slightly higher activity than CYP154C3-2 for most of the substrates. Oxidizing agents (diacetoxyiodo) benzene (PIDA) and hydrogen peroxide (H2O2) were also utilized to actively support the redox reactions, with optimum conversion achieved at concentrations of 3 mM and 65 mM, respectively. The oxidizing agents affected the product distribution, influencing the type and selectivity of the CYP-catalyzed reaction. Additionally, CYP154C3s also catalyzed the C-C bond cleavage of steroids. Therefore, CYP154C3s may be a good candidate for the production of modified steroids for various biological uses.