Functional characterization of a novel CYP119 variant to explore its biocatalytic potential.

Biocatalysts are increasingly applied in the pharmaceutical and chemical industry. Cytochrome P450 enzymes (P450s) are valuable biocatalysts due to their ability to hydroxylate unactivated carbon atoms using molecular oxygen. P450s catalyze reactions using nicotinamide adenine dinucleotide phosphate (NAD(P)H) cofactor and electron transfer proteins. Alternatively, P450s can utilize hydrogen peroxide (H2 O2 ) as an oxidant, but this pathway is inefficient. P450s that show higher efficiency with peroxides are sought after in industrial applications. P450s from thermophilic organisms have more potential applications as they are stable toward high temperature, high and low pH, and organic solvents. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius. In our previous study, a novel T213R/T214I (double mutant [DM]) variant of CYP119 was obtained by screening a mutant library for higher peroxidation activity utilizing H2 O2 . Here, we characterized the substrate scope; stability toward peroxides; and temperature and organic solvent tolerance of DM CYP119 to identify its potential as an industrial biocatalyst. DM CYP119 displayed higher stability than wild-type (WT) CYP119 toward organic peroxides. It shows higher peroxidation activity for non-natural substrates and higher affinity for progesterone and other bioactive potential substrates compared to WT CYP119. DM CYP119 emerges as a new biocatalyst with a wide range of potential applications in the pharmaceutical and chemical industry.

Cloning, expression, and characterization of a novel sericin-like protein.

Silk consists of two proteins called fibroin and sericin. While fibroin is used in the textile industry and has various biomaterial applications, sericin has been considered as waste material until recently. Sericin is a multicomponent protein and it has important properties such as biocompatibility, biodegradability, cryoprotectivity, and antioxidant. Sericin from silkworm cocoons can be obtained by chemical, enzymatic, and heat treatment methods. However, sericin obtained with these treatment methods is not of consistent and high quality. Moreover, the exposure of sericin to harsh conditions during extraction leads to inconsistencies in the composition and structure of the sericin obtained. The inconsistencies in sericin structure and composition decrease application of sericin as a biomaterial. Here, we produce a sericin-like protein (Ser4mer) with native sequence of sericin encoding four repeats of the conserved 38 amino acid motif recombinantly in Escherichia coli and characterize its structural properties. Ser4mer protein shows similar structure to native sericin and higher solubility than previously obtained recombinant sericin-like proteins. Recombinant production of a soluble sericin-like protein will significantly expand its applications as a biomaterial. In addition, recombinant production of silk proteins will allow us to understand sequence-structure relationships in these proteins.

Development of an improved Amplex Red peroxidation activity assay for screening cytochrome P450 variants and identification of a novel mutant of the thermophilic CYP119.

Biocatalysts are increasingly utilized in the synthesis of drugs and agrochemicals as an alternative to chemical catalysis. They are preferred in the synthesis of enantiopure products due to their high regioselectivity and enantioselectivity. Cytochrome P450 (P450) oxygenases are valuable biocatalysts, since they catalyze the oxidation of carbon-hydrogen bonds with high efficiency and selectivity. However, practical use of P450s is limited due to their need for expensive cofactors and electron transport partners. P450s can employ hydrogen peroxide (H2O2) as an oxygen and electron donor, but the reaction with H2O2 is inefficient. The development of P450s that can use H2O2 will expand their applications. Here, an assay that utilizes Amplex Red peroxidation, to rapidly screen H2O2-dependent activity of P450 mutants in cell lysate was developed. This assay was employed to identify mutants of CYP119, a thermophilic P450 from Sulfolobus acidocaldarius, with increased peroxidation activity. A mutant library of CYP119 containing substitutions in the heme active site was constructed via combinatorial active-site saturation test and screened for improved activity. Screening of 158 colonies led to five mutants with higher activity. Among improved variants, T213R/T214I was characterized. T213R/T214I exhibited fivefold higher kcat for Amplex Red peroxidation and twofold higher kcat for styrene epoxidation. T213R/T214I showed higher stability towards heme degradation by H2O2. While the Km for H2O2 and styrene were not altered by the mutation, a fourfold decrease in the affinity for another substrate, lauric acid, was observed. In conclusion, Amplex Red peroxidation screening of CYP119 mutants yielded enzymes with increased peroxide-dependent activity.

A novel thermophilic hemoprotein scaffold for rational design of biocatalysts.

Hemoproteins are commonly found in nature, and involved in many important cellular processes such as oxygen transport, electron transfer, and catalysis. Rational design of hemoproteins can not only inspire novel biocatalysts but will also lead to a better understanding of structure-function relationships in native hemoproteins. Here, the heme nitric oxide/oxygen-binding protein from Caldanaerobacter subterraneus subsp. tengcongensis (TtH-NOX) is used as a novel scaffold for oxidation biocatalyst design. We show that signaling protein TtH-NOX can be reengineered to catalyze H2O2 decomposition and oxidation of 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) by H2O2. In addition, the role of the distal tyrosine (Tyr140) in catalysis is investigated. The mutation of Tyr140 to alanine hinders the catalysis of the oxidation reactions. On the other hand, the mutation of Tyr140 to histidine, which is commonly observed in peroxidases, leads to a significant increase of the catalytic activity. Taken together, these results show that, while the distal histidine plays an important role in hemoprotein reactions with H2O2, it is not always essential for oxidation activity. We show that TtH-NOX protein can be used as an alternative scaffold for the design of novel biocatalysts with desired reactivity or functionality. H-NOX proteins are homologous to the nitric oxide sensor soluble guanylate cyclase. Here, we show that the gas sensor protein TtH-NOX shows limited capacity for catalysis of redox reactions and it can be used as a novel scaffold in biocatalysis design.

The Influence of Nitric Oxide on Soluble Guanylate Cyclase Regulation by Nucleotides: ROLE OF THE PSEUDOSYMMETRIC SITE.

Activation of soluble guanylate cyclase (sGC) by the signaling molecule nitric oxide (NO) leads to formation of the second messenger cGMP, which mediates numerous physiological processes. NO activates sGC by binding to the ferrous heme cofactor; the relative amount of NO with respect to sGC heme affects the enzyme activity. ATP can also influence the activity by binding to an allosteric site, most likely the pseudosymmetric site located in the catalytic domain. Here, the role of the pseudosymmetric site on nucleotide regulation was investigated by point mutations at this site. ATP inhibition kinetics of wild type and a pseudosymmetric site (α1-C594A/ß1-D477A) variant of sGC was determined at various levels of NO. Results obtained show that in the presence of less than 1 eq of NO, there appears to be less than complete activation and little change in the nucleotide binding parameters. The most dramatic effects are observed for the addition of excess NO, which results in an increase in the affinity of GTP at the catalytic site and full activation of sGC. The pseudosymmetric site mutation only affected nucleotide affinities in the presence of excess NO; there was a decrease in the affinity for ATP in both the allosteric and catalytic sites. These observations led to a new kinetic model for sGC activity in the presence of excess NO. This model revealed that the active and allosteric sites show cooperativity. This new comprehensive model gives a more accurate description of sGC regulation by NO and nucleotides in vivo.

Small angle X-ray scattering analysis of thermophilic cytochrome P450 CYP119 and the effects of the N-terminal histidine tag.

Combining size exclusion chromatography-small angle X-ray scattering (SEC-SAXS) and molecular dynamics (MD) analysis is a promising approach to investigate protein behavior in solution, particularly for understanding conformational changes due to substrate binding in cytochrome P450s (CYPs). This study investigates conformational changes in CYP119, a thermophilic CYP from Sulfolobus acidocaldarius that exhibits structural flexibility similar to mammalian CYPs. Although the crystal structure of ligand-free (open state) and ligand-bound (closed state) forms of CYP119 is known, the overall structure of the enzyme in solution has not been explored until now. It was found that theoretical scattering profiles from the crystal structures of CYP119 did not align with the SAXS data, but conformers from MD simulations, particularly starting from the open state (46 % of all frames), agreed well. Interestingly, a small percentage of closed-state conformers also fit the data (9 %), suggesting ligand-free CYP119 samples ligand-bound conformations. Ab initio SAXS models for N-His tagged CYP119 revealed a tail-like unfolded structure impacting protein flexibility, which was confirmed by in silico modeling. SEC-SAXS analysis of N-His CYP119 indicated pentameric structures in addition to monomers in solution, affecting the stability and activity of the enzyme. This study adds insights into the conformational dynamics of CYP119 in solution.

Rational design of thermophilic CYP119 for progesterone hydroxylation by in silico mutagenesis and docking screening.

Steroid-based chemicals can affect the metabolism, immune functions, and development of sexual characteristics. Because of these effects, steroid derivatives are widely used in the pharmaceutical industry. Progesterone is a steroid-based hormone that mainly controls the ovulation period of women but is also a precursor molecule for the synthesis of important hormones like testosterone and cortisone. Cytochrome P450 (CYP) enzymes are important for the production of hydroxyprogesterones in the industry since they can catalyze regio- and enantioselective hydroxylation reactions. Although human CYP enzymes can catalyze hydroxyprogesterone synthesis with high selectivity, these enzymes are membrane bound, which limits their application for industrial production. CYP119 is a soluble and thermophilic enzyme from the archaea Sulfolobus acidocaldarius. Even though the native substrate of the enzyme is not known, CYP119 can catalyze styrene epoxidation, lauric acid hydroxylation, and Amplex®Red peroxidation. In this work, an in silico mutagenesis approach was used to design CYP119 mutants with high progesterone affinity. Energy scores of progesterone docking simulations were used for the design and elimination of single, double, and triple mutants of CYP119. Among designed 674 mutants, five of them match the criteria for progesterone hydroxylation. The most common mutation of these five mutants, L69G mutant was analyzed using independent molecular dynamics (MD) simulations in comparison with the wild-type (WT) enzyme. L69G CYP119, was expressed and isolated from Escherichia coli; it showed 800-fold higher affinity for progesterone compared to WT CYP119. L69G CYP119 also catalyzed progesterone hydroxylation. The novel designed enzyme L69G CYP119 is a potential versatile biocatalyst for progesterone hydroxylation that is expected to be stable under industrial production conditions.

Insight into the rescue of oxidized soluble guanylate cyclase by the activator cinaciguat.

Nitric oxide (NO) signaling mediates many important physiological processes through the receptor soluble guanylate cyclase (sGC). Under disease conditions sGC heme can be oxidized resulting in NO insensitivity. Here, we show that the therapeutic compound cinaciguat (Cin) rescues dysfunctional sGC by direct displacement of the oxidized heme.

Effects of N-Terminal and C-Terminal Polyhistidine Tag on the Stability and Function of the Thermophilic P450 CYP119.

Biocatalysts are sought-after in synthesis of pharmaceuticals and agrochemicals due to their high regioselectivity and enantioselectivity. Among biocatalysts, heme-containing cytochrome P450 (P450) oxygenases are an attractive target since they catalyze oxidation of "unactivated" carbon-hydrogen bonds with high efficiency. CYP119 is an acidothermophilic P450 from Sulfolobus acidocaldarius, which has the potential to be widely used as a biocatalyst since it shows activity at high temperatures and low pH. Polyhistidine tags (His-tags) are widely used to simplify purification of proteins. However, His-tags can cause changes to protein structure and function. Here, we demonstrate the effects of His-tags on CYP119. To this end, the His-tags were cloned at the N-terminus or C-terminus of the CYP119, and His-tagged proteins were expressed and isolated. The thermostability and peroxidase activity of His-tagged CYP119s were tested and compared to wild type CYP119. Results indicated that while addition of His-tags increased the yield and simplified isolation of CYP119, they also influenced the electronic structure of active site and the activity of the protein. We show that N-terminal His-tagged CYP119 has desirable properties and potential to be used in industrial applications, but mechanistic studies using this protein need careful interpretation since the His-tag affects electronic properties of the active site heme iron.

α1-A680T variant in GUCY1A3 as a candidate conferring protection from pulmonary hypertension among Kyrgyz highlanders.

BACKGROUND: Human variation in susceptibility to hypoxia-induced pulmonary hypertension is well recognized. High-altitude residents who do not develop pulmonary hypertension may host protective gene mutations. METHODS AND RESULTS: Exome sequencing was conducted on 24 unrelated Kyrgyz highlanders living 2400 to 3800 m above sea level, 12 (10 men; mean age, 54 years) with an elevated mean pulmonary artery pressure (mean±SD, 38.7±2.7 mm Hg) and 12 (11 men; mean age, 52 years) with a normal mean pulmonary artery pressure (19.2±0.6 mm Hg) to identify candidate genes that may influence the pulmonary vascular response to hypoxia. A total of 140 789 exomic variants were identified and 26 116 (18.5%) were classified as novel or rare. Thirty-three novel or rare potential pathogenic variants (frameshift, essential splice-site, and nonsynonymous) were found exclusively in either ≥3 subjects with high-altitude pulmonary hypertension or ≥3 highlanders with a normal mean pulmonary artery pressure. A novel missense mutation in GUCY1A3 in 3 subjects with a normal mean pulmonary artery pressure encodes an α1-A680T soluble guanylate cyclase (sGC) variant. Expression of the α1-A680T sGC variant in reporter cells resulted in higher cyclic guanosine monophosphate production compared with the wild-type enzyme and the purified α1-A680T sGC exhibited enhanced sensitivity to nitric oxide in vitro. CONCLUSIONS: The α1-A680T sGC variant may contribute to protection against high-altitude pulmonary hypertension and supports sGC as a pharmacological target for reducing pulmonary artery pressure in humans at altitude.

Single-site mutation and secondary structure stability: an isodesmic reaction approach. The case of unnatural amino acid mutagenesis Ala-->Lac.

A method is described to evaluate backbone interactions in proteins via computational unnatural amino acid mutagenesis. Several N-acetyl polyalanyl amides (AcA(n)NH(2)) were optimized in the representative helical (3(10)-, 4(13)-, and a "hybrid" kappa-helix, n = 7, 9, 10, 14) and hairpin (two- and three-stranded antiparallel beta-sheets with type I turns betaalphaalphaepsilon, n = 6, 9, 10) conformations, and extended conformers of N-acetyl polyalanyl methylamides (n = 2, 3) were used to derive multistranded beta-sheet fragments. Subsequently, each residue of every model structure was substituted, one at a time, with l-lactic acid. The resulting mutant structures were again optimized, and group-transfer energies DeltaE(GT) were obtained as heats of the isodesmic reactions: AcA(n)NHR + AcOMe --> AcA(x)LacA(y)NHR + AcNHMe (R = H, CH(3)). These group-transfer energies correlate with the degree of charge polarization of the substituted peptide linkages as measured by the difference Deltae in H and O Mulliken populations in HN-C=O and with the H-bond distances in the "wild-type" structures. A good correlation obtains for the HF/3-21G and B3LYP/6-31G* group-transfer energies. The destabilization effects are interpreted in terms of loss of interstrand and intrastrand H-bonds, decrease in Lewis basicity of the C=O group, and O...O repulsion. On the basis of several comparisons of Ala --> Lac DeltaE(GT)'s with heats of the NH --> CH(2) substitutions, the latter contribution is estimated (B3LYP/6-31G*) to range between 1.5 and 2.4 kcal mol(-1), a figure close to the recent experimental DeltaDeltaG(o) value of 2.6 kcal mol(-1) (McComas, C. C.; Crowley, B. M.; Boger, D. L. J. Am.Chem. Soc. 2003, 125, 9314). The partitioning yields the following maximum values of the electronic association energy of H-bonds in the examined sample of model structures (B3LYP/6-31G* estimates): 3(10)-helix D(e) = -1.7 kcal mol(-1), alpha-helix D(e) = -3.8 kcal mol(-1), beta-sheet D(e) = -6.1 kcal mol(-1). The premise of experimental evaluations of the backbone-backbone H-bonding that Ala --> Lac substitution in proteins is isosteric (e.g., Koh, J. T.; Cornish, V. W.; Schultz, P. G. Biochemistry 1997, 36, 11314) is often but not always corroborated. Examination of the integrity of H-bonding pattern and phi(i), psi(i) distribution identified several mutants with significant distortions of the "wild-type" structure resulting inter alia from the transitions between i, i + 3 and i, i + 4 H-bonding in helices, observed previously in the crystallographic studies of depsipeptides (Ohyama, T.; Oku, H.; Hiroki, A.; Maekawa, Y.; Yoshida, M.; Katakai, R. Biopolymers 2000, 54, 375; Karle, I. L.; Das, C.; Balaram, P. Biopolymers 2001, 59, 276). Thus, the isodesmic reaction approach provides a simple way to gauge how conformation of the polypeptide chain and dimensions of the H-bonding network affect the strength of backbone-backbone C=O...HN bonds. The results indicate that the stabilization provided by such interactions increases on going from 3(10)-helix to alpha-helix to beta-sheet.