Transcriptomic Analysis of the Combined Effects of Methyl Jasmonate and Wounding on Flavonoid and Anthraquinone Biosynthesis in Senna tora.

Jasmonates, including jasmonic acid (JA) and its derivatives such as methyl jasmonate (MeJA) or jasmonly isoleucine (JA-Ile), regulate plant responses to various biotic and abiotic stresses. In this study, we applied exogenous MeJA onto Senna tora leaves subjected to wounding and conducted a transcriptome deep sequencing analysis at 1 (T1), 3 (T3), 6 (T6), and 24 (T24) h after MeJA induction, along with the pretreatment control at 0 h (T0). Out of 18,883 mapped genes, we identified 10,048 differentially expressed genes (DEGs) between the T0 time point and at least one of the four treatment times. We detected the most DEGs at T3, followed by T6, T1, and T24. We observed the upregulation of genes related to JA biosynthesis upon exogenous MeJA application. Similarly, transcript levels of genes related to flavonoid biosynthesis increased after MeJA application and tended to reach their maximum at T6. In agreement, the flavonols kaempferol and quercetin reached their highest accumulation at T24, whereas the levels of the anthraquinones aloe-emodin, emodin, and citreorosein remained constant until T24. This study highlights an increase in flavonoid biosynthesis following both MeJA application and mechanical wounding, whereas no significant influence is observed on anthraquinone biosynthesis. These results provide insights into the distinct regulatory pathways of flavonoid and anthraquinone biosynthesis in response to MeJA and mechanical wounding.

In Silico Analysis and Biochemical Characterization of Streptomyces PET Hydrolase with Bis(2-Hydroxyethyl) Terephthalate Biodegradation Activity.

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.

Structure, dimeric conformation, and coenzyme versatility of p-hydroxybenzoate hydroxylase from Arthrobacter sp. PAMC25564.

p-Hydroxybenzoate hydroxylase (PHBH) catalyzes the ortho-hydroxylation of 4-hydroxybenzoate (4-HB) to protocatechuate (PCA). PHBHs are commonly known as homodimers, and the prediction of pyridine nucleotide binding and specificity remains an ongoing focus in this field. Therefore, our study aimed to determine the dimerization interface in AspPHBH from Arthrobacter sp. PAMC25564 and identify the canonical pyridine nucleotide-binding residues, along with coenzyme specificity, through site-directed mutagenesis. The results confirm a functional dimeric assembly from a tetramer that appeared in the crystallographic asymmetric unit identical to that established in previous studies. Furthermore, AspPHBH exhibits coenzyme versatility, utilizing both NADH and NADPH, with a preference for NADH. Rational engineering experiments demonstrated that targeted mutations in coenzyme surrounding residues profoundly impact NADPH binding, leading to nearly abrogated enzymatic activity compared to that of NADH. R50, R273, and S166 emerged as significant residues for NAD(P)H binding, having a near-fatal impact on NADPH binding compared to NADH. Likewise, the E44 residue plays a critical role in determining coenzyme specificity. Overall, our findings contribute to the fundamental understanding of the determinants of PHBH's active dimeric conformation, coenzyme binding and specificity holding promise for biotechnological advancements.

Exploration of novel trehalases from cold-adapted Variovorax sp. PAMC28711: Functional characterization.

The cold-adapted bacterium Variovorax sp. PAMC28711 possesses two distinct glycoside hydrolase (GH) families of trehalase, GH15 and GH37. While numerous studies have explored bacterial trehalase, the presence of two different trehalase genes within a single strain has not been reported until now. Interestingly, despite both GH37 and GH15 trehalases serving the same purpose of degrading trehalose, but do not share the sequence similarity. The substrate specificity assay confirmed that Vtre37 and Vtre15 displayed hydrolytic activity on α, α-trehalose. The key catalytic sites were identified as D280 and E469 in Vtre37 and E389 and E554 in Vtre15 through site-directed mutation and confirmed these two enzymes belong to trehalase. In addition, Vtre37 exhibited a relatively high level of enzyme activity of 1306.33 (±53.091) µmolmg-1, whereas Vtre15 showed enzyme activity of 408.39 (±12.503) µmolmg-1. Moreover, Vtre37 performed admirably showing resistance to ethanol (10 %), with high stable at acidic pH range. Furthermore, both prediction and experimental results indicate that validoxylamine A showed a potent inhibitory activity against Vtre37 trehalase with a Ki value of 16.85 nM. Therefore, we postulate that Vtre37 could be utilized as an ethanol enhancer and designed for screening inhibitors related to the trehalose degradation pathway. Additionally, we believe that characterizing these bacterial trehalase contributes to a better understanding of trehalose metabolism and its biological importance in bacteria.

Self-clustered GAN for precipitation nowcasting.

This paper proposes a novel GAN framework with self-clustering approach for precipitation nowcasting (ClusterCast). Previous studies have primarily captured the motion vector using only a single latent space, making the models difficult to adapt to disparate space-time distribution of precipitation. Environmental factors (e.g., regional characteristics and precipitation scale) have an impact on precipitation systems and can cause non-stationary distribution. To tackle this problem, our key idea is to train a generator network to predict future radar frames by learning a sub-network that automatically labels precipitation types from a generative model. The training process consists of (i) clustering the hierarchical features derived from the generator stem using a sub-network and (ii) predicting future radar frames according to the self-supervised labels, enabling heterogeneous latent representation. Additionally, we attempt an ensemble forecast that prescribes random perturbations to improve performance. With the flexibility of representation learning, ClusterCast enables the model to learn precipitation distribution more accurately. Results indicate that our method generates non-blurry future frames by preventing mode collapse, and the proposed method demonstrates robustness across various precipitation scenarios. Extensive experiments demonstrate that our method outperforms four benchmarks on a 2-h prediction basis with a mean squared error (MSE) of 8.9% on unseen datasets.

Genomic insight into O-demethylation of 4-methoxybenzoate by a two-component system from Amycolatopsis magusensis KCCM40447.

Cytochrome P450 monooxygenases perform a multitude of roles, including the generation of hydroxylated aromatic compounds that might be utilized by microorganisms for their survival. WGS data of Amycolatopsis magusensis KCCM40447 revealed a complete circular genome of 9,099,986 base pairs and functionally assigned 8601 protein-encoding genes. Genomic analysis confirmed that the gene for 4-methoxybenzoate monoxygenase (CYP199A35) was conserved in close proximity to the gene for 4-hydroxybenzoate transporter (PcaK). The co-localized genes encoding CYP199A35, and ferredoxin-NAD(P) reductase (Mbr) represent a two-component system for electron transfer. CYP199A35 was specific for O-demethylation of para O-methyl substituted benzoic acid derivatives, 4-methoxybenzoate (4 MB), and 4-methoxycinnamic acid (4MCA) using the native redox partner (Mbr); two-component system and non-physiological redox partners (Pdr/Pdx); three-component system. The catalytic efficiency for O-demethylation of 4 MB using Mbr and Pdr/Pdx was 0.02 ± 0.006 min-1 µM-1 and 0.07 ± 0.02 min-1 µM-1 respectively. Further, sequence annotation and function prediction by RAST and KEEG analysis revealed a complete catabolic pathway for the utilization of 4 MB by strain KCCM40447, which was also proved experimentally.

Comparative Genome Analysis of Polar Mesorhizobium sp. PAMC28654 to Gain Insight into Tolerance to Salinity and Trace Element Stress.

In this study, Mesorhizobium sp. PAMC28654 was isolated from a soil sample collected from the polar region of Uganda. Whole-genome sequencing and comparative genomics were performed to better understand the genomic features necessary for Mesorhizobium sp. PAMC28654 to survive and thrive in extreme conditions and stresses. Additionally, diverse sequence analysis tools were employed for genomic investigation. The results of the analysis were then validated using wet-lab experiments. Genome analysis showed trace elements' resistant proteins (CopC, CopD, CzcD, and Acr3), exopolysaccharide (EPS)-producing proteins (ExoF and ExoQ), and nitrogen metabolic proteins (NarG, NarH, and NarI). The strain was positive for nitrate reduction. It was tolerant to 100 mM NaCl at 15 °C and 25 °C temperatures and resistant to multiple trace elements (up to 1 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 1 mM ZnSO4·7H2O, 0.05 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 15 °C and 0.25 mM CuSO4·5H2O, 2 mM CoCl2·6H2O, 0.5 mM ZnSO4·7H2O, 0.01 mM Cd(NO3)2·4H2O, and 100 mM Na2HAsO4·7H2O at 25 °C). This research contributes to our understanding of bacteria's ability to survive abiotic stresses. The isolated strain can be a potential candidate for implementation for environmental and agricultural purposes.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

Analysis of critical residues for peroxygenation and improved peroxygenase activity via in situ H2O2 generation in CYP105D18.

Limited numbers of CYPs have been reported to work naturally as peroxygenases. The peroxide shunt pathway can be efficiently used as an alternative for the NAD(P)H and reductase systems, particularly in high hydrogen peroxide (H2O2) resistance CYPs. We reported the structural and biochemical features of CYP105D18 peroxygenase for its high H2O2 tolerance capacity. Q348 was a crucial residue for the stability of CYP105D18 during the exposure to H2O2. In addition, the role of the hydrophilic amino acid T239 from the I helix for peroxygenation and regiospecificity toward testosterone was investigated. Interestingly, T239E differs in product formation from wild type, catalyzing testosterone to androstenedione in the presence of H2O2. The other variant, T239A, worked with the Pdx/Pdr system and was unable to catalyze testosterone conversion in the presence of H2O2, suggesting the transformation of peroxygenase into monooxygenase. CYP105D18 supported the alternative method of H2O2 used for the catalysis of testosterone. The use of the same concentration of urea hydrogen peroxide adducts in place of direct H2O2 was more efficient for 2ß-hydroxytestosterone conversion. Furthermore, in situ H2O2 generation using GOx/glucose system enhanced the catalytic efficiency (kcat/Km) for wild type and F184A by 1.3- and 1.9-fold, respectively, compared to direct use of H2O2 The engineering of CYP105D18, its improved peroxygenase activity, and alteration in the product oxidation facilitate CYP105D18 as a potential candidate for biotechnological applications.

Elucidation of cold adaptation in Glaciimonas sp. PAMC28666 with special focus on trehalose biosynthesis.

Glaciimonas sp. PAMC28666, an extremophilic bacterium thriving in Antarctic soil and belonging to the Oxalobacteraceae family, represents the only complete genome of its genus available in the NCBI database. Its genome measures 5.2 Mb and comprises 4,476 genes (4,350 protein-coding and 72 non-coding). Phylogenetic analysis shows the strain PAMC28666 in a unique branch within the genus Glaciimonas, closely related to Glaciimonas alpine Cr9-12, supported by robust bootstrap values. In addition, strain PAMC28666 showed 77.08 and 23.3% ANI and DDH, respectively, with Glaciimonas sp. PCH181.This study focuses on how polar strain PAMC28666 responds to freeze-thaw conditions, Experimental results revealed a notable survival rate of 47.28% when subjected to a temperature of 15°C for a period of 10 days. Notably, two genes known to be responsive to cold stress, Trehalose 6-phosphate synthase (otsA) and Trehalose 6-phosphate phosphatase (otsB), exhibited increased expression levels as the temperature shifted from 25°C to 15°C. The upregulation of otsAB and the consequent synthesis of trehalose play pivotal roles in enhancing the cold resistance of strain PAMC28666, offering valuable insights into the correlation between trehalose production and adaptation to cold stress. Furthermore, research into this neglected cold-adapted variation, like Glaciimonas sp. PAMC28666, has the potential to shed light on how trehalose is produced in cold-adapted environments Additionally, there is potential to extract trehalose compounds from this strain for diverse biotechnological applications, including food and cosmetics, with ongoing research exploring its unique properties.

Evidential deep learning for trustworthy prediction of enzyme commission number.

The rapid growth of uncharacterized enzymes and their functional diversity urge accurate and trustworthy computational functional annotation tools. However, current state-of-the-art models lack trustworthiness on the prediction of the multilabel classification problem with thousands of classes. Here, we demonstrate that a novel evidential deep learning model (named ECPICK) makes trustworthy predictions of enzyme commission (EC) numbers with data-driven domain-relevant evidence, which results in significantly enhanced predictive power and the capability to discover potential new motif sites. ECPICK learns complex sequential patterns of amino acids and their hierarchical structures from 20 million enzyme data. ECPICK identifies significant amino acids that contribute to the prediction without multiple sequence alignment. Our intensive assessment showed not only outstanding enhancement of predictive performance on the largest databases of Uniprot, Protein Data Bank (PDB) and Kyoto Encyclopedia of Genes and Genomes (KEGG), but also a capability to discover new motif sites in microorganisms. ECPICK is a reliable EC number prediction tool to identify protein functions of an increasing number of uncharacterized enzymes.

ResBiGAAT: Residual Bi-GRU with attention for protein-ligand binding affinity prediction.

Protein-ligand interaction plays a crucial role in drug discovery, facilitating efficient drug development and enabling drug repurposing. Several computational algorithms, such as Graph Neural Networks and Convolutional Neural Networks, have been proposed to predict the binding affinity using the three-dimensional structure of ligands and proteins. However, there are limitations due to the need for experimental characterization of the three-dimensional structure of protein sequences, which is still lacking for some proteins. Moreover, these models often suffer from unnecessary complexity, resulting in extraneous computations. This study presents ResBiGAAT, a novel deep learning model that combines a deep Residual Bidirectional Gated Recurrent Unit with two-sided self-attention mechanisms. ResBiGAAT leverages protein and ligand sequence-level features and their physicochemical properties to efficiently predict protein-ligand binding affinity. Through rigorous evaluation using 5-fold cross-validation, we demonstrate the performance of our proposed approach. The model exhibits competitive performance on an external dataset, highlighting its generalizability. Our publicly available web interface, located at resbigaat.streamlit.app, allows users to conveniently input protein and ligand sequences to estimate binding affinity.

Biochemical and structural basis of mercuric reductase, GbsMerA, from Gelidibacter salicanalis PAMC21136.

Heavy metals, including mercury, are non-biodegradable and highly toxic to microorganisms even at low concentrations. Understanding the mechanisms underlying the environmental adaptability of microorganisms with Hg resistance holds promise for their use in Hg bioremediation. We characterized GbsMerA, a mercury reductase belonging to the mercury-resistant operon of Gelidibacter salicanalis PAMC21136, and found its maximum activity of 474.7 µmol/min/mg in reducing Hg+2. In the presence of Ag and Mn, the enzyme exhibited moderate activity as 236.5 µmol/min/mg and 69 µmol/min/mg, respectively. GbsMerA exhibited optimal activity at pH 7.0 and a temperature of 60 °C. Moreover, the crystal structure of GbsMerA and structural comparison with homologues indicated that GbsMerA contains residues, Tyr437´ and Asp47, which may be responsible for metal transfer at the si-face by providing a hydroxyl group (-OH) to abstract a proton from the thiol group of cysteine. The complex structure with NADPH indicated that Y174 in the re-face can change its side chain direction upon NADPH binding, indicating that Y174 may have a role as a gate for NADPH binding. Moreover, the heterologous host expressing GbsMerA (pGbsMerA) is more resistant to Hg toxicity when compared to the host lacking GbsMerA. Overall, this study provides a background for understanding the catalytic mechanism and Hg detoxification by GbsMerA and suggests the application of genetically engineered E. coli strains for environmental Hg removal.

Identification and evaluation of CAZyme genes, along with functional characterization of a new GH46 chitosanase from Streptomyces sp. KCCM12257.

The genomic analysis of Streptomyces sp. KCCM12257 presented 233 CAZyme genes with a predominant glycosyl hydrolase family. This contributes degradation of various polysaccharides including chitin and chitosan, and other promising candidates for the production of different oligosaccharides. We screened the strain providing different polysaccharides as a sole source of carbon and strain KCCM12257, showed higher activity towards colloidal chitosan. Further, we identified and characterized a new chitosanase (MDI5907146) of GH46 family. There was no activity towards chitin, carboxymethylcellulose, or even with chitosan powder. This enzyme acts on colloidal chitosan and hydrolyzes it down into monoacetyl chitobiose, which consists of two glucosamine units with an acetyl group attached to them. The maximum enzyme activity was observed at pH 6.5 and 40 °C using colloidal chitosan as a substrate. The Co2+ metal ions almost double the reaction as compared to other metal ions. The dissociation constant (Km) and of colloidal chitosan (≥90 % and ≥75%DD) were 3.03 mg/ml and 5.01 mg/ml respectively, while maximum velocity (Vmax) values were found to be 36 mg/ml, and 30 µM/µg/min, respectively. Similarly, catalytic efficiency (Kcat/Km) of colloidal chitosan with ≥90 %DD was 1.9 fold higher than colloidal chitosan with ≥75%DD.

Comparative Genomic Analysis of Biofilm-Forming Polar Microbacterium sp. Strains PAMC22086 and PAMC21962 Isolated from Extreme Habitats.

The members of Microbacterium isolated from different environments are known to form peptidoglycan. In this study, we compared the biofilm-forming abilities of Microbacterium sp. PAMC22086 (PAMC22086), which was isolated from the soil in the South Shetland Islands and Microbacterium sp. PAMC21962 (PAMC21962), which was isolated from algae in the South Shetland Islands. The analysis of average nucleotide identity and phylogeny of PAMC22086 revealed a 97% similarity to Microbacterium oxydans VIU2A, while PAMC21962 showed a 99.1% similarity to Microbacterium hominis SGAir0570. For the comparative genomic analysis of PAMC22086 and PAMC21962, the genes related to biofilm formation were identified using EggNOG and KEGG pathway databases. The genes possessed by both PAMC22086 and PAMC21962 are cpdA, phnB, rhlC, and glgC, which regulate virulence, biofilm formation, and multicellular structure. Among the genes indirectly involved in biofilm formation, unlike PAMC21962, PAMC22086 possessed csrA, glgC, and glgB, which are responsible for attachment and glycogen biosynthesis. Additionally, in PAMC22086, additional functional genes rsmA, which is involved in mobility and polysaccharide production, and dksA, GTPase, and oxyR, which play roles in cell cycle and stress response, were identified. In addition, the biofilm-forming ability of the two isolates was examined in vivo using the standard crystal violet staining technique, and morphological differences in the biofilm were investigated. It is evident from the different distribution of biofilm-associated genes between the two strains that the bacteria can survive in different niches by employing distinct strategies. Both strains exhibit distinct morphologies. PAMC22086 forms a biofilm that attaches to the side, while PAMC21962 indicates growth starting from the center. The biofilm formation-related genes in Microbacterium are not well understood. However, it has been observed that Microbacterium species form biofilm regardless of the number of genes they possess. Through comparison between different Microbacterium species, it was revealed that specific core genes are involved in cell adhesion, which plays a crucial role in biofilm formation. This study provides a comprehensive profile of the Microbacterium genus's genomic features and a preliminary understanding of biofilm in this genus, laying the foundation for further research.

Complete Genome Analysis of Subtercola sp. PAMC28395: Genomic Insights into Its Potential Role for Cold Adaptation and Biotechnological Applications.

This study reports the complete genome sequence of Subtercola sp. PAMC28395, a strain isolated from cryoconite in Uganda. This strain possesses several active carbohydrate-active enzyme (CAZyme) genes involved in glycogen and trehalose metabolism. Additionally, two specific genes associated with α-galactosidase (GH36) and bacterial alpha-1,2-mannosidase (GH92) were identified in this strain. The presence of these genes indicates the likelihood that they can be expressed, enabling the strain to break down specific polysaccharides derived from plants or the shells of nearby crabs. The authors performed a comparative analysis of CAZyme patterns and biosynthetic gene clusters (BGCs) in several Subtercola strains and provided annotations describing the unique characteristics of these strains. The comparative analysis of BGCs revealed that four strains, including PAMC28395, have oligosaccharide BGCs, and we confirmed that the pentose phosphate pathway was configured perfectly in the genome of PAMC28395, which may be associated with adaptation to low temperatures. Additionally, all strains contained antibiotic resistance genes, indicating a complex self-resistance system. These results suggest that PAMC28395 can adapt quickly to the cold environment and produce energy autonomously. This study provides valuable information on novel functional enzymes, particularly CAZymes, that operate at low temperatures and can be used for biotechnological applications and fundamental research purposes.

Crystal Structure and Biochemical Analysis of a Cytochrome P450 Steroid Hydroxylase (BaCYP106A6) from Bacillus Species.

Cytochrome P450 (CYP) is a heme-containing enzyme that catalyzes hydroxylation reactions with various substrate molecules. Steroid hydroxylases are particularly useful for effectively introducing hydroxyl groups into a wide range of steroids in the pharmaceutical industry. This study reports a newly identified CYP steroid hydroxylase (BaCYP106A6) from the bacterium Bacillus sp. and characterizes it using an in vitro enzyme assay and structural investigation. Bioconversion assays indicated that BaCYP106A1 catalyzes the hydroxylation of progesterone and androstenedione, whereas no or low conversion was observed with 11ß-hydroxysteroids such as cortisol, corticosterone, dexamethasone, and prednisolone. In addition, the crystal structure of BaCYP106A6 was determined at a resolution of 2.8 Å to investigate the configuration of the substrate-binding site and understand substrate preference. This structural characterization and comparison with other bacterial steroid hydroxylase CYPs allowed us to identify a unique Arg295 residue that may serve as the key residue for substrate specificity and regioselectivity in BaCYP106A6. This observation provides valuable background for further protein engineering to design commercially useful CYP steroid hydroxylases with different substrate specificities.

Complete Genome Sequence of the Antibiotic-Resistant Pseudomonas fluorescens Strain Ant01, from the Rhizosphere of Antarctic Moss.

Pseudomonas fluorescens Ant01 was isolated as an antibiotic-resistant strain from the rhizosphere of a moss from Barton Peninsula, King George Island, Antarctica. The assembled genome size is 6,249,144 bp, with 5,616 protein-coding genes, 69 tRNA genes, and 19 rRNA genes.

H2O2-Driven Hydroxylation of Steroids Catalyzed by Cytochrome P450 CYP105D18: Exploration of the Substrate Access Channel.

CYP105D18 supports H2O2 as an oxygen surrogate for catalysis well and shows high H2O2 resistance capacity. We report the hydroxylation of different steroids using H2O2 as a cosubstrate. Testosterone was regiospecifically hydroxylated to 2ß-hydroxytestosterone. Based on the experimental data and molecular docking, we predicted that hydroxylation of methyl testosterone and nandrolone would occur at position 2 in the A-ring, while hydroxylation of androstenedione and adrenosterone was predicted to occur in the B-ring. Further, structure-guided rational design of the substrate access channel was performed with the mutagenesis of residues S63, R82, and F184. Among the mutants, S63A showed a marked decrease in product formation, while F184A showed a significant increase in product formation in testosterone, nandrolone, methyl testosterone, androstenedione, and adrenosterone. The catalytic efficiency (kcat/Km) toward testosterone was increased 1.36-fold in the F184A mutant over that in the wild-type enzyme. These findings might facilitate the potential use of CYP105D18 and further engineering to establish the basis of biotechnological applications. IMPORTANCE The structural modification of steroids is a challenging chemical reaction. Modifying the core ring and the side chain improves the biological activity of steroids. In particular, bacterial cytochrome P450s are used as promiscuous enzymes for the activation of nonreactive carbons of steroids. In the present work, we reported the H2O2-mediated hydroxylation of steroids by CYP105D18, which also overcomes the use of expensive cofactors. Further, exploring the substrate access channel and modifying the bulky amino acid F184A increase substrate conversion while modifying the substrate recognizing amino acid S63 markedly decreases product formation. Exploring the substrate access channel and the rational design of CYP105D18 can improve the substrate conversion, which facilitates the engineering of P450s for industrial application.