Crystal structure of a transcription factor, GerE (PaGerE), from spore-forming bacterium Paenisporosarcina sp. TG-14.

In cold and harsh environments such as glaciers and sediments in ice cores, microbes can survive by forming spores. Spores are composed of a thick coat protein, which protects against external factors such as heat-shock, high salinity, and nutrient deficiency. GerE is a key transcription factor involved in spore coat protein expression in the mother cell during sporulation. GerE regulates transcription during the late sporulation stage by directly binding to the promoter of cotB gene. Here, we report the crystal structure of PaGerE at 2.09 Šresolution from Paenisporosarcina sp. TG-14, which was isolated from the Taylor glacier. The PaGerE structure is composed of four α-helices and adopts a helix-turn-helix architecture with 68 amino acid residues. Based on our DNA binding analysis, the PaGerE binds to the promoter region of CotB to affect protein expression. Additionally, our structural comparison studies suggest that DNA binding by PaGerE causes a conformational change in the α4-helix region, which may strongly induce dimerization of PaGerE.

Structural and functional analysis of a dimeric fumarylacetoacetate hydrolase (EaFAH) from psychrophilic Exiguobacterium antarcticum.

Fumarylacetoacetate hydrolase (FAH) is essential for the degradation of aromatic amino acids as well as for the cleavage of carbon-carbon bonds in metabolites or small organic compounds. Here, the X-ray crystal structure of EaFAH, a dimeric fumarylacetoacetate hydrolase from Exiguobacterium antarcticum, was determined, and its functional properties were investigated using biochemical methods. EaFAH adopts a mixed ß-sandwich roll fold with a highly flexible lid region (Val73-Leu94), and an Mg2+ ion is bound at the active site by coordinating to the three carboxylate oxygen atoms of Glu124, Glu126, and Asp155. The hydrolytic activity of EaFAH toward various substrates, including linalyl acetate was investigated using native polyacrylamide gel electrophoresis, activity staining, gel filtration, circular dichroism spectroscopy, fluorescence, and enzyme assays.

Structural and functional characterization of a novel cold-active S-formylglutathione hydrolase (SfSFGH) homolog from Shewanella frigidimarina, a psychrophilic bacterium.

BACKGROUND: S-Formylglutathione is hydrolyzed to glutathione and formate by an S-formylglutathione hydrolase (SFGH) (3.1.2.12). This thiol esterase belongs to the esterase family and is also known as esterase D. SFGHs contain highly conserved active residues of Ser-Asp-His as a catalytic triad at the active site. Characterization and investigation of SFGH from Antarctic organisms at the molecular level is needed for industrial use through protein engineering. RESULTS: A novel cold-active S-formylglutathione hydrolase (SfSFGH) from Shewanella frigidimarina, composed of 279 amino acids with a molecular mass of ~ 31.0 kDa, was characterized. Sequence analysis of SfSFGH revealed a conserved pentapeptide of G-X-S-X-G found in various lipolytic enzymes along with a putative catalytic triad of Ser148-Asp224-His257. Activity analysis showed that SfSFGH was active towards short-chain esters, such as p-nitrophenyl acetate, butyrate, hexanoate, and octanoate. The optimum pH for enzymatic activity was slightly alkaline (pH 8.0). To investigate the active site configuration of SfSFGH, we determined the crystal structure of SfSFGH at 2.32 Å resolution. Structural analysis shows that a Trp182 residue is located at the active site entrance, allowing it to act as a gatekeeper residue to control substrate binding to SfSFGH. Moreover, SfSFGH displayed more than 50% of its initial activity in the presence of various chemicals, including 30% EtOH, 1% Triton X-100, 1% SDS, and 5 M urea. CONCLUSIONS: Mutation of Trp182 to Ala allowed SfSFGH to accommodate a longer chain of substrates. It is thought that the W182A mutation increases the substrate-binding pocket and decreases the steric effect for larger substrates in SfSFGH. Consequently, the W182A mutant has a broader substrate specificity compared to wild-type SfSFGH. Taken together, this study provides useful structure-function data of a SFGH family member and may inform protein engineering strategies for industrial applications of SfSFGH.

Multidimensional Anodized Titanium Foam Photoelectrode for Efficient Utilization of Photons in Mesoscopic Solar Cells.

Mesoscopic solar cells based on nanostructured oxide semiconductors are considered as a promising candidates to replace conventional photovoltaics employing costly materials. However, their overall performances are below the sufficient level required for practical usages. Herein, this study proposes an anodized Ti foam (ATF) with multidimensional and hierarchical architecture as a highly efficient photoelectrode for the generation of a large photocurrent. ATF photoelectrodes prepared by electrochemical anodization of freeze-cast Ti foams have three favorable characteristics: (i) large surface area for enhanced light harvesting, (ii) 1D semiconductor structure for facilitated charge collection, and (iii) 3D highly conductive metallic current collector that enables exclusion of transparent conducting oxide substrate. Based on these advantages, when ATF is utilized in dye-sensitized solar cells, short-circuit photocurrent density up to 22.0 mA cm-2 is achieved in the conventional N719 dye-I3- /I- redox electrolyte system even with an intrinsically inferior quasi-solid electrolyte.

Peroxide-dependent oxidation reactions catalyzed by CYP191A1 from Mycobacterium smegmatis.

OBJECTIVES: To find the catalytic activities of CYP191A1 from Mycobacterium smegmatis, in which functions of most P450s are unknown, by using a set of reductase systems, peroxides, and various substrates including fatty acids and human drugs. RESULTS: CYP191A1 was functionally expressed in Escherichia coli and purified. Its catalytic activities were examined with fatty acids, chromogenic and fluorogenic substrates, and several human P450 substrates, in the presence of six different types of electron transfer systems, such as rat NADPH-P450 reductase, Candida NADPH-P450 reductase, ferredoxin/ferredoxin reductase, putidaredoxin/putidaredoxin reductase, and peroxides (H2O2 and t-butyl hydroperoxide). The reactions catalyzed by CYP191A1 included the hydroxylation and O-dealkylation of several substrates. CONCLUSIONS: CYP191A1 preferentially catalyzes the peroxide-dependent oxidation of various substrates over the reductase-dependent reaction. Its peroxygenase activity may be used an effective biocatalytic tool to synthesize the metabolites of drugs.

Marine Antifreeze Proteins: Structure, Function, and Application to Cryopreservation as a Potential Cryoprotectant.

Antifreeze proteins (AFPs) are biological antifreezes with unique properties, including thermal hysteresis(TH),ice recrystallization inhibition(IRI),and interaction with membranes and/or membrane proteins. These properties have been utilized in the preservation of biological samples at low temperatures. Here, we review the structure and function of marine-derived AFPs, including moderately active fish AFPs and hyperactive polar AFPs. We also survey previous and current reports of cryopreservation using AFPs. Cryopreserved biological samples are relatively diverse ranging from diatoms and reproductive cells to embryos and organs. Cryopreserved biological samples mainly originate from mammals. Most cryopreservation trials using marine-derived AFPs have demonstrated that addition of AFPs can improve post-thaw viability regardless of freezing method (slow-freezing or vitrification), storage temperature, and types of biological sample type.

Engineering Synthetic Multistress Tolerance in Escherichia coli by Using a Deinococcal Response Regulator, DR1558.

Cellular robustness is an important trait for industrial microbes, because the microbial strains are exposed to a multitude of different stresses during industrial processes, such as fermentation. Thus, engineering robustness in an organism in order to push the strains toward maximizing yield has become a significant topic of research. We introduced the deinococcal response regulator DR1558 into Escherichia coli (strain Ec-1558), thereby conferring tolerance to hydrogen peroxide (H2O2). The reactive oxygen species (ROS) level in strain Ec-1558 was reduced due to the increased KatE catalase activity. Among four regulators of the oxidative-stress response, OxyR, RpoS, SoxS, and Fur, we found that the expression of rpoS increased in Ec-1558, and we confirmed this increase by Western blot analysis. Electrophoretic mobility shift assays showed that DR1558 bound to the rpoS promoter. Because the alternative sigma factor RpoS regulates various stress resistance-related genes, we performed stress survival analysis using an rpoS mutant strain. Ec-1558 was able to tolerate a low pH, a high temperature, and high NaCl concentrations in addition to H2O2, and the multistress tolerance phenotype disappeared in the absence of rpoS. Microarray analysis clearly showed that a variety of stress-responsive genes that are directly or indirectly controlled by RpoS were upregulated in strain Ec-1558. These findings, taken together, indicate that the multistress tolerance conferred by DR1558 is likely routed through RpoS. In the present study, we propose a novel strategy of employing an exogenous response regulator from polyextremophiles for strain improvement.

Crystal Structure of a Putative Cytochrome P450 Alkane Hydroxylase (CYP153D17) from Sphingomonas sp. PAMC 26605 and Its Conformational Substrate Binding.

Enzymatic alkane hydroxylation reactions are useful for producing pharmaceutical and agricultural chemical intermediates from hydrocarbons. Several cytochrome P450 enzymes catalyze the regio- and stereo-specific hydroxylation of alkanes. We evaluated the substrate binding of a putative CYP alkane hydroxylase (CYP153D17) from the bacterium Sphingomonas sp. PAMC 26605. Substrate affinities to C10-C12 n-alkanes and C10-C14 fatty acids with Kd values varied from 0.42 to 0.59 µM. A longer alkane (C12) bound more strongly than a shorter alkane (C10), while shorter fatty acids (C10, capric acid; C12, lauric acid) bound more strongly than a longer fatty acid (C14, myristic acid). These data displayed a broad substrate specificity of CYP153D17, hence it was named as a putative CYP alkane hydroxylase. Moreover, the crystal structure of CYP153D17 was determined at 3.1 Å resolution. This is the first study to provide structural information for the CYP153D family. Structural analysis showed that a co-purified alkane-like compound bound near the active-site heme group. The alkane-like substrate is in the hydrophobic pocket containing Thr74, Met90, Ala175, Ile240, Leu241, Val244, Leu292, Met295, and Phe393. Comparison with other CYP structures suggested that conformational changes in the ß1-ß2, α3-α4, and α6-α7 connecting loop are important for incorporating the long hydrophobic alkane-like substrate. These results improve the understanding of the catalytic mechanism of CYP153D17 and provide valuable information for future protein engineering studies.

Regioselective hydroxylation of omeprazole enantiomers by bacterial CYP102A1 mutants.

A large set of Bacillus megaterium CYP102A1 mutants are known to metabolize various drugs to form human metabolites. Omeprazole (OMP), a proton pump inhibitor, has been widely used as an acid inhibitory agent for the treatment of gastric acid hypersecretion disorders. It is primarily metabolized by human CYP2C19 and CYP3A4 to 5'-OH OMP and a sulfone product, respectively. It was recently reported that several CYP102A1 mutants can oxidize racemic and S-OMP to 5'-OH OMP and that these mutants can further oxidize 5'-OH racemic OMP to 5'-COOH OMP. Here, we report that the S- and R-enantiomers of OMP are hydroxylated by 26 mutants of CYP102A1 to produce 1 major metabolite (5'-OH OMP) regardless of the chirality of the parent substrates. Although the binding of R-OMP to the CYP102A1 active site caused a more apparent change of heme environment compared with binding of S-OMP, there was no correlation between the spectral change upon substrate binding and catalytic activity of either enantiomer. The 5'-OH OMP produced from racemic, S-, and R-OMP could be obtained with a high conversion rate and high selectivity when the triple R47L/F87V/L188Q mutant was used. These results suggest that bacterial CYP102A1 mutants can be used to produce the human metabolite 5'-OH OMP from both the S- and R-enantiomers of OMP.

Chimeric cytochromes P450 engineered by domain swapping and random mutagenesis for producing human metabolites of drugs.

Human drug metabolites produced by cytochrome P450 enzymes are critical for safety testing and may themselves act as drugs or leads in the drug discovery and development process. Here, highly active chimeric fusion proteins (chimeras) were obtained by reductase domain swapping of mutants at key catalytic residues of the heme domain with that of a natural variant (CYP102A1.2) of P450 BM3 (CYP102A1.1) from Bacillus megaterium. Random mutagenesis at the heme domain of the chimera was also used to generate chimeric mutants that were more active and diverse than the chimeras themselves. To determine whether the chimeras and several mutants of the highly active chimera displayed enhanced catalytic activity and, more importantly, whether they acquired activities of biotechnological importance, we measured the oxidation activities of the chimeras and chimeric mutants toward human P450 substrates, mainly drugs. Some of the chimeric mutants showed high activity toward typical human P450 substrates including drugs. Statin leads, especially chiral products, with inhibitory effects toward HMG-CoA reductase could be obtained from metabolites of statin drugs generated using these chimeric mutants. This study reveals the critical role of the reductase domain for the activity of P450 BM3 and shows that chimeras generated by domain swapping can be used to develop industrial enzymes for the synthesis of human metabolites from drugs and drug leads.

Highly ordered and vertically oriented TiO2/Al2O3 nanotube electrodes for application in dye-sensitized solar cells.

The surface of long TiO2 nanotube (NT) electrodes in dye-sensitized solar cells (DSSCs) was modified without post-annealing by using atomic layer deposition (ALD) for the enhancement of photovoltage. Vertically oriented TiO2 NT electrodes with highly ordered and crack-free surface structures over large areas were prepared by a two-step anodization method. The prepared TiO2 NTs had a pore size of 80 nm, and a length of 23 µm. Onto these TiO2 NTs, an Al2O3 shell of a precisely controlled thickness was deposited by ALD. The conformally coated shell layer was confirmed by high-resolution transmission electron microscopy, energy-dispersive x-ray spectroscopy, and x-ray photoelectron spectroscopy. The open-circuit voltage (V(oc)) of the DSSCs was gradually enhanced as the thickness of the Al2O3 shell of the TiO2/Al2O3 NT electrodes was increased, which resulted from the enhanced electron lifetime. The enhanced electron lifetime caused by the energy barrier effect of the shell layer was measured quantitatively by the open-circuit voltage decay technique. As a result, 1- and 2-cycle-coated samples showed enhanced conversion efficiencies compared to the bare sample.

Predicting CYP2C19 catalytic parameters for enantioselective oxidations using artificial neural networks and a chirality code.

Cytochromes P450 (CYP for isoforms) play a central role in biological processes especially metabolism of chiral molecules; thus, development of computational methods to predict parameters for chiral reactions is important for advancing this field. In this study, we identified the most optimal artificial neural networks using conformation-independent chirality codes to predict CYP2C19 catalytic parameters for enantioselective reactions. Optimization of the neural networks required identifying the most suitable representation of structure among a diverse array of training substrates, normalizing distribution of the corresponding catalytic parameters (k(cat), K(m), and k(cat)/K(m)), and determining the best topology for networks to make predictions. Among different structural descriptors, the use of partial atomic charges according to the CHelpG scheme and inclusion of hydrogens yielded the most optimal artificial neural networks. Their training also required resolution of poorly distributed output catalytic parameters using a Box-Cox transformation. End point leave-one-out cross correlations of the best neural networks revealed that predictions for individual catalytic parameters (k(cat) and K(m)) were more consistent with experimental values than those for catalytic efficiency (k(cat)/K(m)). Lastly, neural networks predicted correctly enantioselectivity and comparable catalytic parameters measured in this study for previously uncharacterized CYP2C19 substrates, R- and S-propranolol. Taken together, these seminal computational studies for CYP2C19 are the first to predict all catalytic parameters for enantioselective reactions using artificial neural networks and thus provide a foundation for expanding the prediction of cytochrome P450 reactions to chiral drugs, pollutants, and other biologically active compounds.

Generation of human chiral metabolites of simvastatin and lovastatin by bacterial CYP102A1 mutants.

Recently, the wild-type and mutant forms of cytochrome P450 BM3 (CYP102A1) from Bacillus megaterium were found to oxidize various xenobiotic substrates, including pharmaceuticals, of human P450 enzymes. Simvastatin and lovastatin, which are used to treat hyperlipidemia and hypercholesterolemia, are oxidized by human CYP3A4/5 to produce several metabolites, including 6'ß-hydroxy (OH), 3″-OH, and exomethylene products. In this report, we show that the oxidation of simvastatin and lovastatin was catalyzed by wild-type CYP102A1 and a set of its mutants, which were generated by site-directed and random mutagenesis. One major hydroxylated product (6'ß-OH) and one minor product (6'-exomethylene), but not other products, were produced by CYP102A1 mutants. Formation of the metabolites was confirmed by high-performance liquid chromatography, liquid chromatography-mass spectroscopy, and NMR. Chemical methods to synthesize the metabolites of simvastatin and lovastatin have not been reported. These results demonstrate that CYP102A1 mutants can be used to produce human metabolites, especially chiral metabolites, of simvastatin and lovastatin. Our computational findings suggest that a conformational change in the cavity of the mutant active sites is related to the activity change. The modeling results also suggest that the activity change results from the movement of several specific residues in the active sites of the mutants. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward simvastatin and lovastatin.

Structural Insights into a Bifunctional Peptide Methionine Sulfoxide Reductase MsrA/B Fusion Protein from Helicobacter pylori.

Methionine sulfoxide reductase (Msr) is a family of enzymes that reduces oxidized methionine and plays an important role in the survival of bacteria under oxidative stress conditions. MsrA and MsrB exist in a fusion protein form (MsrAB) in some pathogenic bacteria, such as Helicobacter pylori (Hp), Streptococcus pneumoniae, and Treponema denticola. To understand the fused form instead of the separated enzyme at the molecular level, we determined the crystal structure of HpMsrABC44S/C318S at 2.2 Å, which showed that a linker region (Hpiloop, 193-205) between two domains interacted with each HpMsrA or HpMsrB domain via three salt bridges (E193-K107, D197-R103, and K200-D339). Two acetate molecules in the active site pocket showed an sp2 planar electron density map in the crystal structure, which interacted with the conserved residues in fusion MsrABs from the pathogen. Biochemical and kinetic analyses revealed that Hpiloop is required to increase the catalytic efficiency of HpMsrAB. Two salt bridge mutants (D193A and E199A) were located at the entrance or tailgate of Hpiloop. Therefore, the linker region of the MsrAB fusion enzyme plays a key role in the structural stability and catalytic efficiency and provides a better understanding of why MsrAB exists in a fused form.

Structural basis of the cooperative activation of type II citrate synthase (HyCS) from Hymenobacter sp. PAMC 26554.

Citrate synthase (CS) catalyzes the formation of citrate and coenzyme A from acetyl-CoA and oxaloacetate. CS exists in two forms: type I and type II. We determined the citrate-bound crystal structure of type II CS from the Hymenobacter sp. PAMC 26554 bacterium (HyCS; isolated from Antarctic lichen). Citrate molecules bound to a cleft between the large and small domains of HyCS. Structural comparison of HyCS with other type II CSs revealed that type II CSs have a highly conserved flexible hinge region (residues G264-P265 in HyCS), enabling correct positioning of active site residues. Notably, the catalytic His266 residue of HyCS interacted with Trp262 in the inactive (unliganded open) state of other type II CSs, whereas the His266 residue moved to the active site via a small-domain swing motion, interacting with the bound citrate in the closed conformation of HyCS. However, type I CSs lack this tryptophan residue and face-to-edge interactions. Thus, type II CSs might have a unique domain-motion control mechanism enabling a tight allosteric regulation. An activity assay using a W262A mutant showed a Hill coefficient of 2.4; thus, the interaction between Trp262 and His266 was closely related to the positive cooperative ligand binding of type II CS.

Crystal structure of a MarR family protein from the psychrophilic bacterium Paenisporosarcina sp. TG-14 in complex with a lipid-like molecule.

MarR family proteins regulate the transcription of multiple antibiotic-resistance genes and are widely found in bacteria and archaea. Recently, a new MarR family gene was identified by genome analysis of the psychrophilic bacterium Paenisporosarcina sp. TG-14, which was isolated from sediment-laden basal ice in Antarctica. In this study, the crystal structure of the MarR protein from Paenisporosarcina sp. TG-14 (PaMarR) was determined at 1.6 Šresolution. In the crystal structure, a novel lipid-type compound (palmitic acid) was found in a deep cavity, which was assumed to be an effector-binding site. Comparative structural analysis of homologous MarR family proteins from a mesophile and a hyperthermophile showed that the DNA-binding domain of PaMarR exhibited relatively high mobility, with a disordered region between the ß1 and ß2 strands. In addition, structural comparison with other homologous complex structures suggests that this structure constitutes a conformer transformed by palmitic acid. Biochemical analysis also demonstrated that PaMarR binds to cognate DNA, where PaMarR is known to recognize two putative binding sites depending on its molar concentration, indicating that PaMarR binds to its cognate DNA in a stoichiometric manner. The present study provides structural information on the cold-adaptive MarR protein with an aliphatic compound as its putative effector, extending the scope of MarR family protein research.

Engineering bacterial cytochrome P450 (P450) BM3 into a prototype with human P450 enzyme activity using indigo formation.

Human cytochrome P450 (P450) enzymes metabolize a variety of endogenous and xenobiotic compounds, including steroids, drugs, and environmental chemicals. In this study, we examine the possibility that bacterial P450 BM3 (CYP102A1) mutants with indole oxidation activity have the catalytic activities of human P450 enzymes. Error-prone polymerase chain reaction was carried out on the heme domain-coding region of the wild-type gene to generate a CYP102A1 DNA library. The library was transformed into Escherichia coli for expression of the P450 mutants. A colorimetric colony-based method was adopted for primary screening of the mutants. When the P450 activities were measured at the whole-cell level, some of the blue colonies, but not the white colonies, possessed apparent oxidation activity toward coumarin and 7-ethoxycoumarin, which are typical human P450 substrates that produce fluorescent products. Coumarin is oxidized by the CYP102A1 mutants to produce two metabolites, 7-hydroxycoumarin and 3-hydroxycoumarin. In addition, 7-ethoxycoumarin is simultaneously oxidized to 7-hydroxycoumarin by O-deethylation reaction and to 3-hydroxy,7-ethoxycoumarin by 3-hydroxylation reactions. Highly active mutants are also able to metabolize several other human P450 substrates, including phenacetin, ethoxyresorufin, and chlorzoxazone. These results indicate that indigo formation provides a simple assay for identifying CYP102A1 mutants with a greater potential for human P450 activity. Furthermore, our computational findings suggest a correlation between the stabilization of the binding site and the catalytic efficiency of CYP102A1 mutants toward coumarin: the more stable the structure in the binding site, the lower the energy barrier and the higher the catalytic efficiency.

Structural basis of small RNA hydrolysis by oligoribonuclease (CpsORN) from Colwellia psychrerythraea strain 34H.

Cells regulate their intracellular mRNA levels by using specific ribonucleases. Oligoribonuclease (ORN) is a 3'-5' exoribonuclease for small RNA molecules, important in RNA degradation and re-utilisation. However, there is no structural information on the ligand-binding form of ORNs. In this study, the crystal structures of oligoribonuclease from Colwellia psychrerythraea strain 34H (CpsORN) were determined in four different forms: unliganded-structure, thymidine 5'-monophosphate p-nitrophenyl ester (pNP-TMP)-bound, two separated uridine-bound, and two linked uridine (U-U)-bound forms. The crystal structures show that CpsORN is a tight dimer, with two separated active sites and one divalent metal cation ion in each active site. These structures represent several snapshots of the enzymatic reaction process, which allowed us to suggest a possible one-metal-dependent reaction mechanism for CpsORN. Moreover, the biochemical data support our suggested mechanism and identified the key residues responsible for enzymatic catalysis of CpsORN.

Crystal Structure and Functional Characterization of a Xylose Isomerase (PbXI) from the Psychrophilic Soil Microorganism, Paenibacillus sp.

Xylose isomerase (XI; E.C. 5.3.1.5) catalyzes the isomerization of xylose to xylulose, which can be used to produce bioethanol through fermentation. Therefore, XI has recently gained attention as a key catalyst in the bioenergy industry. Here, we identified, purified, and characterized a XI (PbXI) from the psychrophilic soil microorganism, Paenibacillus sp. R4. Surprisingly, activity assay results showed that PbXI is not a cold-active enzyme, but displays optimal activity at 60°C. We solved the crystal structure of PbXI at 1.94-Å resolution to investigate the origin of its thermostability. The PbXI structure shows a (ß/α)8-barrel fold with tight tetrameric interactions and it has three divalent metal ions (CaI, CaII, and CaIII). Two metal ions (CaI and CaII) located in the active site are known to be involved in the enzymatic reaction. The third metal ion (CaIII), located near the ß4-α6 loop region, was newly identified and is thought to be important for the stability of PbXI. Compared with previously determined thermostable and mesophilic XI structures, the ß1-α2 loop structures near the substrate binding pocket of PbXI were remarkably different. Site-directed mutagenesis studies suggested that the flexible ß1-α2 loop region is essential for PbXI activity. Our findings provide valuable insights that can be applied in protein engineering to generate lowtemperature purpose-specific XI enzymes.

Characterization of two steroid hydroxylases from different Streptomyces spp. and their ligand-bound and -unbound crystal structures.

Bacterial cytochrome P450 (CYP) enzymes are involved in the hydroxylation of various endogenous substrates while using a heme molecule as a cofactor. CYPs have gained biotechnological interest as useful biocatalysts capable of altering chemical structures by adding a hydroxyl group in a regiospecific manner. Here, we identified, purified, and characterized two CYP154C4 proteins from Streptomyces sp. W2061 (StCYP154C4-1) and Streptomyces sp. ATCC 11861 (StCYP154C4-2). Activity assays showed that both StCYP154C4-1 and StCYP154C4-2 can produce 2'-hydroxylated testosterone, which differs from the activity of a previously described NfCYP154C5 from Nocardia farcinica in terms of its 16α-hydroxylation of testosterone. To better understand the molecular basis of the regioselectivity of these two CYP154C4 proteins, crystal structures of the ligand-unbound form of StCYP154C4-1 and the testosterone-bound form of StCYP154C4-2 were determined. Comparison with the previously determined NfCYP154C5 structure revealed differences in the substrate-binding residues, suggesting a likely explanation for the different patterns of testosterone hydroxylation, despite the high sequence similarities between the enzymes (54% identity). These findings provide valuable insights that will enable protein engineering for the development of artificial steroid-related CYPs exhibiting different regiospecificity.