Biocatalyst Engineering by Assembly of Fatty Acid Transport and Oxidation Activities for In Vivo Application of Cytochrome P-450BM-3 Monooxygenase

The application of whole cells containing cytochrome P-450_BM-3 monooxygenase [EC 1.14.14.1] for the bioconversion of long-chain saturated fatty acids to ω-1, ω-2, and ω-3 hydroxy fatty acids was investigated. We utilized pentadecanoic acid and studied its conversion to a mixture of 12-, 13-, and 14-hydroxypentadecanoic acids by this monooxygenase. For this purpose, Escherichia coli recombinants containing plasmid pCYP102 producing the fatty acid monooxygenase cytochrome P-450_BM-3 were used. To overcome inefficient uptake of pentadecanoic acid by intact E. coli cells, we made use of a cloned fatty acid uptake system from Pseudomonas oleovorans which, in contrast to the common FadL fatty acid uptake system of E. coli, does not require coupling by FadD (acyl-coenzyme A synthetase) of the imported fatty acid to coenzyme A. This system from P. oleovorans is encoded by a gene carried by plasmid pGEc47, which has been shown to effect facilitated uptake of oleic acid in E. coli W3110 (M. Nieboer, Ph.D. thesis, University of Groningen, Groningen, The Netherlands, 1996). By using a double recombinant of E. coli K27, which is a fadD mutant and therefore unable to consume substrates or products via the β-oxidation cycle, a twofold increase in productivity was achieved. Applying cytochrome P-450_BM-3 monooxygenase as a biocatalyst in whole cells does not require the exogenous addition of the costly cofactor NADPH. In combination with the coenzyme A-independent fatty acid uptake system from P. oleovorans, cytochrome P-450_BM-3 recombinants appear to be useful alternatives to the enzymatic approach for the bioconversion of long-chain fatty acids to subterminal hydroxylated fatty acids.Cytochrome P-450_BM-3 monooxygenase (CytP450_BM-3) is a soluble NADPH-dependent monooxygenase from Bacillus megaterium ATCC 14581 (13). It is a class II P-450 enzyme that contains flavin adenine dinucleotide, flavin mononucleotide, and a heme moiety (17). Unlike most CytP450 monooxygenases, which consist of a distinct monooxygenase and a reductase, CytP450_BM-3 contains these functionalities in a single polypeptide (3, 15, 18).The enzyme hydroxylates a variety of long-chain aliphatic substrates, such as fatty acids, alkanols, and alkylamides at the ω-1, ω-2, and ω-3 positions (4, 17), and oxidizes unsaturated fatty acids to epoxides in vitro (17, 23) with high enantioselectivity. Oxidation of eicosapentenoic acid (C_20:5) and arachidonic acid (C_20:4) yielded 17(S),18(R)-epoxyeicosatetraenoic acid (94% enantiomeric excess [e.e.]) for the former and a mixture of 18-(R)-hydroxyarachidonic acid (92% e.e.) and 14(S),15(R)-epoxyeicosatrienoic acid at 98% e.e. for the latter substrate (8). Recently, it has been demonstrated that the enzyme also produces α,ω diacids from ω-oxo fatty acids by oxidation of the terminal aldehyde functionality (9). The catalytic constant (k_cat) of CytP450_BM-3 is among the highest found for P-450 monooxygenases, ranging from 15 s⁻¹ for laureate to 75 s⁻¹ for pentadecanoic acid (11). For comparison, a typical microsomal P-450 monooxygenase from human liver (CYP2J2) had a k_cat of 10⁻³ s⁻¹ for arachidonic acid (32), compared to a k_cat of 55 s⁻¹ for CytP450_BM-3 for the same substrate (8).This high catalytic efficiency prompted us to investigate the applicability of CytP450_BM-3 as a biocatalyst for the subterminal hydroxylation of long-chain fatty acids (LCFAs). Since these subterminal hydroxy LCFAs are chiral molecules, their application in the production of enantiopure synthetic building blocks, especially for pharmaceutical agents, could be envisioned. Further, long-chain hydroxy acids find applications as precursors for polymers or cyclic lactones, which are used as components of fragrances and as antibiotics. Although chemical syntheses have been developed for ω-1 hydroxy fatty acids (from C₁₂ to C₁₈) (26, 28, 29) and for ω-2 and ω-3 hydroxyoctadecanoic acids (2), they require expensive functionalized substrates and are in general complicated, multistep processes (26, 28, 29) which cannot be carried out with unmodified fatty acids as inexpensive starting material. In principle, such inexpensive substrates can be oxidized to hydroxy fatty acids by biocatalysts, either in vitro or in vivo. The latter is preferred, since whole cells actively regenerate the NADPH required for fatty acid oxidation with monooxygenases such as CytP450_BM-3. In designing a suitable whole-cell biocatalyst, several additional points had to be considered.First, uptake must be efficient. Second, degradation of substrate or product must be avoided. In fact, biotransformations of fatty acids with whole cells are usually inefficient due to limited uptake of these compounds at neutral pH, and when taken up, they are degraded via β-oxidation. The transport of LCFAs in Escherichia coli is mediated via the fadL and fadD gene products. FadL is the transporter that carries LCFAs across the outer membrane and is absolutely required for LCFA transport (20). FadD, the acyl coenzyme A (CoA) synthetase, is located at the inner side of the cytoplasmic membrane and is required for formation of the acyl coenzyme A thioester, after which the activated fatty acids are channeled into the β-oxidation cycle for fatty acid degradation (21, 22). Thus, we used a FadD mutant, E. coli K27, as a suitable host for the production of subterminal hydroxyalkanoic acids (20). E. coli K27 cannot couple free fatty acids to coenzyme A, thus preventing substrate or product degradation by the host. Such fadD mutants are, however, also impaired in efficient uptake of fatty acids (20). We circumvented this by introducing a fatty acid uptake system from Pseudomonas oleovorans encoded on pGEc47. Finally, we introduced the P-450_BM-3 monooxygenase on pCYP102 into the fadD mutant E. coli. The resulting recombinant, E. coli K27(pCYP102, pGEc47), is a promising tailored biocatalyst for the oxidation of saturated LCFAs to ω-1, ω-2, and ω-3 hydroxy fatty acids.     

Non-Antigen-Specific B-Cell Activation following Murine Gammaherpesvirus Infection Is CD4 Independent In Vitro but CD4 Dependent In Vivo

The murine gammaherpesvirus MHV-68 multiplies in the respiratory epithelium after intranasal inoculation, then spreads to infect B cells in lymphoid germinal centers. Exposing B cells to MHV-68 in vitro caused an increase in cell size, up-regulation of the CD69 activation marker, and immunoglobulin M (IgM) production. The infectious process in vivo was also associated with increased CD69 expression on B cells in the draining lymph nodes and spleen, together with a rise in total serum Ig. However, whereas the in vitro effect on B cells was entirely T-cell independent, evidence of in vivo B-cell activation was minimal in CD4⁺ T-cell-deficient (I-A^b−/−) or CD4⁺ T-cell-depleted mice. Furthermore, the Ig present at high levels in serum was predominantly of the IgG class. Surprisingly, the titer of influenza virus-specific serum IgG in previously immunized mice fell following MHV-68 infection, suggesting that there was relatively little activation of memory B cells. Thus, CD4⁺ T cells seemed both to amplify a direct viral activation of B cells in lymphoid tissue and to promote new Ig class switching despite a lack of obvious cognate antigen.Herpesvirus (HV) infections are often associated with non-antigen-specific B-cell activation (13, 14, 16, 21, 22). Although no definite role has been established for this process in viral pathogenesis, it is of particular interest in gammaherpesvirus (γ-HV) infections, since chronic B-cell stimulation may contribute to the oncogenesis (9, 15) associated with Epstein-Barr virus (EBV) and human herpesvirus 8 (HHV-8) infections. Infection with EBV activates B cells expressing the immunoglobulin (Ig) V_4-34 gene (4), which is also overrepresented in certain lymphomas (6, 25). EBV-activated V_4-34-expressing B cells can undergo somatic mutation and isotype switching, indicating a participation in normal germinal-center interactions (5). The latent membrane protein 1 (LMP-1) of EBV, which has intracellular signaling substrates similar to those of CD40 (12), and LMP-2A, which can trigger lymphocyte activation (2), may both contribute to this process. However, analysis of lymphocyte interactions in vivo has not been possible with the human γ-HVs.The murine γ-HV-68 (MHV-68) is a natural γ-HV of small rodents that is related to EBV (8) and to HHV-8 (33). After intranasal (i.n.) infection of conventional mice, the virus spreads from the lung to the lymphoid tissue (29) and then persists in B lymphocytes (28) and in epithelial cells (27). This persistent infection is associated with an infectious mononucleosis-like illness (7, 20) characterized by a CD4-dependent splenomegaly and an increase in viral load (31). In BALB/c mice, MHV-68 causes an acute and apparently non-antigen-specific rise in total serum IgG (26). The virus-specific serum antibody response is, in contrast, relatively slow in onset and does not reach plateau levels until 2 to 3 months after infection (26). MHV-68-infected C57BL/6J (B6) mice have more IgG⁺ cells and fewer IgM⁺ cells in the spleen (18) than uninfected controls, but to what extent this represents normal immunity is unclear.There is evidence (3) of MHV-68 infection in splenic germinal centers, and both the non-antigen-specific B-cell activation and the CD4-dependent increase in viral load may reflect an exploitation by the virus of normal germinal-center function. The present analysis defines the need, or lack thereof, for CD4⁺ T-cell help to drive B-cell activation following in vitro or in vivo exposure to MHV-68.     

Identification of CRM1-dependent Nuclear Export Cargos Using Quantitative Mass Spectrometry

Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the major transport receptor for the export of proteins from the nucleus. It binds to nuclear export signals (NESs) that are rich in leucines and other hydrophobic amino acids. The prediction of NESs is difficult because of the extreme recognition flexibility of CRM1. Furthermore, proteins can be exported upon binding to an NES-containing adaptor protein. Here we present an approach for identifying targets of the CRM1-export pathway via quantitative mass spectrometry using stable isotope labeling with amino acids in cell culture. With this approach, we identified >100 proteins from HeLa cells that were depleted from cytosolic fractions and/or enriched in nuclear fractions in the presence of the selective CRM1-inhibitor leptomycin B. Novel and validated substrates are the polyubiquitin-binding protein sequestosome 1, the cancerous inhibitor of protein phosphatase 2A (PP2A), the guanine nucleotide-binding protein-like 3-like protein, the programmed cell death protein 2-like protein, and the cytosolic carboxypeptidase 1 (CCP1). We identified a functional NES in CCP1 that mediates direct binding to the export receptor CRM1. The method will be applicable to other nucleocytoplasmic transport pathways, as well as to the analysis of nucleocytoplasmic shuttling proteins under different growth conditions.The transport of macromolecules across the nuclear envelope occurs through large proteinaceous structures called nuclear pore complexes (NPCs).¹ NPCs are composed of ∼30 nucleoporins that occur in copy numbers of eight or multiples of eight, leading to a complex with a total size of ∼125 MDa in vertebrate cells (1, 2). Active nucleocytoplasmic transport of proteins is a signal- and energy-dependent process that is mostly mediated by transport receptors of the importin β-superfamily called karyopherins or importins/exportins (3, 4). These proteins interact not only with their cargos, but also with certain nucleoporins, facilitating the translocation of the transport complex across the NPC. For nuclear export, at least seven nuclear export receptors/exportins have been identified (3, 4). Chromosome region maintenance 1/exportin1/Exp1/Xpo1 (CRM1) is the most important export receptor for proteins in yeast and vertebrates, and it is also involved in the export of several RNA species (5). Very little is known about the interaction of CRM1 with nucleoporins. Binding to cargo molecules, in contrast, is very well described. Exported proteins typically carry a nuclear export signal (NES) that is enriched with leucines or other hydrophobic amino acids. Such leucine-rich NESs were first discovered in the HIV type 1 Rev protein (6) and the cAMP-dependent protein kinase inhibitor (7). The consensus sequence consists of four key hydrophobic amino acids (leucine, isoleucine, valine, and phenylalanine or methionine; denoted by Φ¹–Φ⁴) following the sequence Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ₄, with x preferentially being a charged polar or small amino acid (for a review, see Ref. 8). A structural analysis of different NES peptides revealed a fifth hydrophobic amino acid in some substrates involved in CRM1 recognition, leading to a revised consensus sequence of Φ⁰-(x)-Φ¹-(x)_2–3-Φ²-(x)_2–3-Φ³-(x)-Φ⁴ (9). In a very recent study, Chook and coworkers established a novel database, NESdb, for NES-containing proteins and analyzed the sequence requirements for proteins in that database in detail (10, 11). “Supraphysiological ” substrates with NESs that fulfill all criteria bind CRM1 with very high affinity and can outcompete other substrates (9, 12). Apart from linear sequences, CRM1 might recognize more complex export signals, such as in fatty acid binding protein 4, in which a functional NES is established only in the tertiary structure of the protein (13), or in snurportin 1 (SPN1), in which sequences outside of the NES proper contribute to CRM1 binding (14–16). This high level of complexity in the recognition sequence for the export receptor makes it very difficult to predict potential CRM1-dependent export cargos using bioinformatics tools. Nevertheless, >200 potential CRM substrates have been described so far (11, 17–19; see also NESbase 1.0 and NESdb).The small GTP-binding protein Ran also plays an essential role in CRM1-mediated nuclear export, as it binds cooperatively to the export receptor, together with the NES cargo. As the affinity of many NES substrates for CRM1 is rather low, the formation of this trimeric transport complex seems to be a rate-limiting step in nuclear export (20). On the nuclear side of the NPC, a number of accessory factors such as RanBP3 (21, 22), Nup98 (23), and NLP1 (24) can further promote the formation of export complexes. Following export, RanBP1 and RanGAP initiate the disassembly of the export complex (for a review, see Ref. 3).A powerful tool for the analysis of CRM1-mediated export is the fungal metabolite leptomycin B (LMB). LMB originally was discovered as an antifungal antibiotic in Streptomyces (25) and later turned out to be a specific and selective inhibitor of the CRM1-mediated nuclear export pathway (26, 27). It binds covalently to cysteine 528 in the NES-binding region of human CRM1 (28), preventing the formation of trimeric export complexes (for a review, see Ref. 5).We used a quantitative MS-based approach (stable isotope labeling with amino acids in cell culture (SILAC)) to evaluate the nuclear export characteristics of proteins by measuring changes in their relative abundance in subcellular fractions after blocking the CRM1-mediated nuclear export with LMB. Using this approach, we identified known and novel CRM1-targets and characterized the NES of one cargo, cytosolic carboxypeptidase 1 (CCP1), in detail.     

In Vivo Mutational Analysis of YtvA from Bacillus subtilis: MECHANISM OF LIGHT ACTIVATION OF THE GENERAL STRESS RESPONSE

The general stress response of Bacillus subtilis can be activated by stimuli such as the addition of salt or ethanol and with blue light. In the latter response, YtvA activates σ^B through a cascade of Rsb proteins, organized in stressosomes. YtvA is composed of an N-terminal LOV (light, oxygen, and voltage) domain and a C-terminal STAS (sulfate transporter and anti-sigma factor) domain and shows light-modulated GTP binding in vitro. Here, we examine the mechanism of YtvA-mediated activation of σ^B in vivo with site-directed mutagenesis. Constitutive off and constitutive on mutations have been identified. Disruption of GTP binding in the STAS domain eliminates light activation of σ^B. In contrast, modification of sites relevant for phosphorylation of STAS domains does not affect the stress response significantly. The data obtained are integrated into a model for the structure of full-length YtvA, which presumably functions as a dimer.LOV² domains (1), members of the superfamily of PAS domains (2, 3), are abundant in all domains of life and were first identified in plant phototropins (4). These photoreceptors regulate stomatal opening, phototropism, etc. and contain two N-terminal LOV domains that confer light regulation on the C-terminal Ser/Thr kinase domain (4). They also occur in bacteria, in which YtvA from Bacillus subtilis has been best characterized (for a review, see e.g. Ref. 5). Its N-terminal LOV domain binds FMN and shows the typical LOV photochemistry (6, 7): covalent adduct formation between a cysteine and the FMN chromophore. A linker helix, denoted Jα (7), connects the LOV domain to a STAS domain. The latter domain is present in many regulators of the general stress response of B. subtilis (8, 9). Stress via the addition of salt or ethanol (for a review, see Ref. 10) and blue light (11, 12) activates the general stress response via the environmental pathway, which integrates various signals via a large multiprotein complex, called the stressosome (13, 14). YtvA, which mediates light activation of σ^B (11, 12, 15), co-purifies with other STAS domain proteins in the stressosomes (16).When cells are stressed, STAS domains of several stressosome proteins (e.g. RsbS and RsbR) are phosphorylated by another intrinsic stressosome component, the serine/threonine kinase RsbT (9, 14, 17, 18). Next, RsbT is released from the complex to trigger RsbU, a protein phosphatase, thus (indirectly) activating σ^B (19). Phosphorylation of YtvA, however, has never been detected. Rather, it has been demonstrated in vitro that YtvA shows light-dependent GTP binding, presumably at its NTP-binding site in the STAS domain (20).Little is known about the mechanism of signal transmission in and by YtvA, except that in the C62A mutant, photochemistry in vitro (12) and light activation of σ^B in vivo (12, 15) are abolished. More detailed information is available for LOV domains of phototropins. A conserved glutamine, which is in hydrogen-bonding contact with the isoalloxazine ring of FMN, rotates its side chain by 180° upon covalent adduct formation (21). Replacement of this residue by leucine in the LOV2 domain of Phy3 from Adiantum results in a considerable reduction of the light-induced structural change (22). The corresponding mutation in phototropin 1 from Arabidopsis impairs autophosphorylation activity (23). The signal generated in the LOV2 domain is transmitted to the downstream kinase domain of phototropin 1 of Avena sativa through disruption of the interaction between its central β-sheet and the C-terminal linker region, the Jα-helix (24).Here, we study the mechanism of activation of YtvA in vivo, i.e. light-induced activation of the σ^B response, with site-directed mutagenesis. We focus on three regions of the protein, the flavin-binding pocket, the β-sheet of the LOV domain, and the GTP-binding site, and on potential phosphorylation sites of the STAS domain. We demonstrate that light-activated GTP binding is crucial for functional YtvA. A computational approach was used to model the structure of full-length YtvA. The model suggests that light modulates accessibility of the GTP-binding site of the STAS domain of YtvA.