Redirecting photosynthetic electron flux in the cyanobacterium Synechocystis sp. PCC 6803 by the deletion of flavodiiron protein Flv3.

BACKGROUND: Oxygen-evolving photoautotrophic organisms, like cyanobacteria, protect their photosynthetic machinery by a number of regulatory mechanisms, including alternative electron transfer pathways. Despite the importance in modulating the electron flux distribution between the photosystems, alternative electron transfer routes may compete with the solar-driven production of CO2-derived target chemicals in biotechnological systems under development. This work focused on engineered cyanobacterial Synechocystis sp. PCC 6803 strains, to explore possibilities to rescue excited electrons that would normally be lost to molecular oxygen by an alternative acceptor flavodiiron protein Flv1/3-an enzyme that is natively associated with transfer of electrons from PSI to O2, as part of an acclimation strategy towards varying environmental conditions. RESULTS: The effects of Flv1/3 inactivation by flv3 deletion were studied in respect to three alternative end-products, sucrose, polyhydroxybutyrate and glycogen, while the photosynthetic gas fluxes were monitored by Membrane Inlet Mass Spectrometry (MIMS) to acquire information on cellular carbon uptake, and the production and consumption of O2. The results demonstrated that a significant proportion of the excited electrons derived from photosynthetic water cleavage was lost to molecular oxygen via Flv1/3 in cells grown under high CO2, especially under high light intensities. In flv3 deletion strains these electrons could be re-routed to increase the relative metabolic flux towards the monitored target products, but the carbon distribution and the overall efficiency were determined by the light conditions and the genetic composition of the respective pathways. At the same time, the total photosynthetic capacity of the Δflv3 strains was systematically reduced, and accompanied by upregulation of oxidative glycolytic metabolism in respect to controls with the native Flv1/3 background. CONCLUSIONS: The observed metabolic changes and respective production profiles were proposedly linked with the lack of Flv1/3-mediated electron transfer, and the associated decrease in the intracellular ATP/NADPH ratio, which is bound to affect the metabolic carbon partitioning in the flv3-deficient cells. While the deletion of flv3 could offer a strategy for enhancing the photosynthetic production of desired chemicals in cyanobacteria under specified conditions, the engineered target pathways have to be carefully selected to align with the intracellular redox balance of the cells.

Comparison of orthologous cyanobacterial aldehyde deformylating oxygenases in the production of volatile C3-C7 alkanes in engineered E. coli.

Aldehyde deformylating oxygenase (ADO) is a unique enzyme found exclusively in photosynthetic cyanobacteria, which natively converts acyl aldehyde precursors into hydrocarbon products embedded in cellular lipid bilayers. This capacity has opened doors for potential biotechnological applications aiming at biological production of diesel-range alkanes and alkenes, which are compatible with the nonrenewable petroleum-derived end-products in current use. The development of production platforms, however, has been limited by the relative inefficiency of ADO enzyme, promoting research towards finding new strategies and information to be used for rational design of enhanced pathways for hydrocarbon over-expression. In this work we present an optimized approach to study different ADO orthologs derived from different cyanobacterial species in an in vivo set-up in Escherichia coli. The system enabled comparison of alternative ADOs for the production efficiency of short-chain volatile C3-C7 alkanes, propane, pentane and heptane, and provided insight on the differences in substrate preference, catalytic efficiency and limitations associated with the enzymes. The work concentrated on five ADO orthologs which represent the most extensively studied cyanobacterial species in the field, and revealed distinct differences between the enzymes. In most cases the ADO from Nostoc punctiforme PCC 73102 performed the best in respect to yields and initial rates for the production of the volatile hydrocarbons. At the other extreme, the system harboring the ADO form Synechococcus sp. RS9917 produced very low amounts of the short-chain alkanes, primarily due to poor accumulation of the enzyme in E. coli. The ADOs from Synechocystis sp. PCC 6803 and Prochlorococcus marinus MIT9313, and the corresponding variant A134F displayed less divergence, although variation between chain-length preferences could be observed. The results confirmed the general trend of ADOs having decreasing catalytic efficiency towards precursors of decreasing chain-length, while expanding the knowledge on the species-specific traits, which may aid future pathway design and structure-based engineering of ADO for more efficient hydrocarbon production systems.

Structure-based engineering of angucyclinone 6-ketoreductases.

Angucyclines are tetracyclic polyketides produced by Streptomyces bacteria that exhibit notable biological activities. The great diversity of angucyclinones is generated in tailoring reactions, which modify the common benz[a]anthraquinone carbon skeleton. In particular, the opposite stereochemistry of landomycins and urdamycins/gaudimycins at C-6 is generated by the short-chain alcohol dehydrogenases/reductases LanV and UrdMred/CabV, respectively. Here we present crystal structures of LanV and UrdMred in complex with NADP(+) and the product analog rabelomycin, which enabled us to identify four regions associated with the functional differentiation. The structural analysis was confirmed in chimeragenesis experiments focusing on these regions adjacent to the active site cavity, which led to reversal of the activities of LanV and CabV. The results surprisingly indicated that the conformation of the substrate and the stereochemical outcome of 6-ketoreduction appear to be intimately linked.

Structural and functional analysis of angucycline C-6 ketoreductase LanV involved in landomycin biosynthesis.

Angucyclines are biologically active natural products constructed around a common benz[a]anthraquinone carbon frame. One key branching point in the biosynthesis of angucyclines is the ketoreduction at C-6, which results in the opposite stereochemistry of landomycins and urdamycins/gaudimycins. Here we present the 1.65 Å resolution crystal structure of LanV from Streptomyces cyanogenus S136 that is responsible for the 6R stereochemistry of landomycins. The enzyme displays the common architectural fold of short-chain alcohol dehydrogenases/reductases and contains bound nicotinamide adenine dinucleotide phosphate. Determination of the structure of LanV in complex with 11-deoxylandomycinone at 2.0 Å resolution indicated that substrate binding does not induce large conformational changes and that substrate recognition occurs mainly through hydrophobic interactions. Analysis of the electron density map of the ternary complex revealed that the catalytic reaction had most likely proceeded backward in the crystal, because the data could be best fit with a compound harboring a carbonyl group at C-6. A coordinated water molecule was atypically identified between the ligand and the conserved Tyr160 residue, which was confirmed to be critical for the catalytic activity by site-directed mutagenesis. A catalytic triad of Ser147, Tyr160, and Lys164 could be recognized on the basis of the crystal structure, and stereoselective labeling studies demonstrated that the transfer of hydride from reduced nicotinamide adenine dinucleotide phosphate to the substrate occurs from the 4-pro-S side of the cosubstrate. Importantly, Ser192 was identified as being involved in controlling the stereochemistry of the reaction, as assays with single mutant Ser192Ile led to accumulation of gaudimycin C with 6S stereochemistry as a minor product.

Tracing the evolution of angucyclinone monooxygenases: structural determinants for C-12b hydroxylation and substrate inhibition in PgaE.

Two functionally distinct homologous flavoprotein hydroxylases, PgaE and JadH, have been identified as branching points in the biosynthesis of the polyketide antibiotics gaudimycin C and jadomycin A, respectively. These evolutionarily related enzymes are both bifunctional and able to catalyze the same initial reaction, C-12 hydroxylation of the common angucyclinone intermediate prejadomycin. The enzymes diverge in their secondary activities, which include hydroxylation at C-12b by PgaE and dehydration at C-4a/C-12b by JadH. A further difference is that the C-12 hydroxylation is subject to substrate inhibition only in PgaE. Here we have identified regions associated with the C-12b hydroxylation in PgaE by extensive chimeragenesis, focusing on regions surrounding the active site. The results highlight the importance of a hairpin-ß motif near the dimer interface, with two nonconserved residues, P78 and I79 (corresponding to Q89 and F90, respectively, in JadH), and invariant residue H73 playing key roles. Kinetic characterization of PgaE variants demonstrates that the secondary C-12b hydroxylation and substrate inhibition by prejadomycin are likely to be interlinked. The crystal structure of the PgaE P78Q/I79F variant at 2.4 Å resolution confirms that the changes do not alter the conformation of the ß-strand secondary structure and that the side chains of these residues in effect point away from the active site toward the dimer interface. The results support a catalytic model for PgaE containing two binding modes for C-12 and C-12b hydroxylations, where binding of prejadomycin in the orientation for C-12b hydroxylation leads to substrate inhibition. The presence of an allosteric network is evident based on enzyme kinetics.

Tailoring enzymes involved in the biosynthesis of angucyclines contain latent context-dependent catalytic activities.

Comparison of homologous angucycline modification enzymes from five closely related Streptomyces pathways (pga, cab, jad, urd, lan) allowed us to deduce the biosynthetic steps responsible for the three alternative outcomes: gaudimycin C, dehydrorabelomycin, and 11-deoxylandomycinone. The C-12b-hydroxylated urdamycin and gaudimycin metabolites appear to be the ancestral representatives from which landomycins and jadomysins have evolved as a result of functional divergence of the ketoreductase LanV and hydroxylase JadH, respectively. Specifically, LanV has acquired affinity for an earlier biosynthetic intermediate resulting in a switch in biosynthetic order and lack of hydroxyls at C-4a and C-12b, whereas in JadH, C-4a/C-12b dehydration has evolved into an independent secondary function replacing C-12b hydroxylation. Importantly, the study reveals that many of the modification enzymes carry several alternative, hidden, or ancestral catalytic functions, which are strictly dependent on the biosynthetic context.

Identification of late-stage glycosylation steps in the biosynthetic pathway of the anthracycline nogalamycin.

Nogalamycin is an anthracycline antibiotic that has been shown to exhibit significant cytotoxicity. Its biological activity requires two deoxysugar moieties: nogalose and nogalamine, which are attached at C7 and C1, respectively, of the aromatic polyketide aglycone. Curiously, the aminosugar nogalamine is also connected through a C-C bond between C2 and C5''. Despite extensive molecular genetic characterization of early biosynthetic steps, nogalamycin glycosylation has not been investigated in detail. Here we show that expression of the majority of the gene cluster in Streptomyces albus led to accumulation of three new anthracyclines, which unexpectedly included nogalamycin derivatives in which nogalamine was replaced either by rhodosamine with the C-C bond intact (nogalamycin R) or by 2-deoxyfucose without the C-C bond (nogalamycin F). In addition, a monoglycosylated intermediate-3',4'-demethoxynogalose-1-hydroxynogalamycinone-was isolated. Importantly, when the remaining biosynthetic genes were introduced into the heterologous host by using a two-plasmid system, nogalamycin could be isolated from the cultures, thus indicating that the whole gene cluster had been identified. We further show that one of the three glycosyltransferases (GTs) residing in the cluster-snogZ-appears to be redundant, whereas gene inactivation experiments revealed that snogE and snogD act as nogalose and nogalamine transferases, respectively. The substrate specificity of the nogalamine transferase SnogD was demonstrated in vitro: the enzyme was able to remove 2deoxyfucose from nogalamycin F. All of the new compounds were found to inhibit human topoisomerase I in activity measurements, whereas only nogalamycin R showed minor activity against topoisomerase II.

Structure of collagen receptor integrin α(1)I domain carrying the activating mutation E317A.

We have analyzed the structure and function of the integrin α(1)I domain harboring a gain-of-function mutation E317A. To promote protein crystallization, a double variant with an additional C139S mutation was used. In cell adhesion assays, the E317A mutation promoted binding to collagen. Similarly, the double mutation C139S/E317A increased adhesion compared with C139S alone. Furthermore, soluble α(1)I C139S/E317A was a higher avidity collagen binder than α(1)I C139S, indicating that the double variant represents an activated form. The crystal structure of the activated variant of α(1)I was solved at 1.9 Å resolution. The E317A mutation results in the unwinding of the αC helix, but the metal ion has moved toward loop 1, instead of loop 2 in the open α(2)I. Furthermore, unlike in the closed αI domains, the metal ion is pentacoordinated and, thus, prepared for ligand binding. Helix 7, which has moved downward in the open α(2)I structure, has not changed its position in the activated α(1)I variant. During the integrin activation, Glu(335) on helix 7 binds to the metal ion at the metal ion-dependent adhesion site (MIDAS) of the ß(1) subunit. Interestingly, in our cell adhesion assays E317A could activate collagen binding even after mutating Glu(335). This indicates that the stabilization of helix 7 into its downward position is not required if the α(1) MIDAS is already open. To conclude, the activated α(1)I domain represents a novel conformation of the αI domain, mimicking the structural state where the Arg(287)-Glu(317) ion pair has just broken during the integrin activation.

Flavoprotein hydroxylase PgaE catalyzes two consecutive oxygen-dependent tailoring reactions in angucycline biosynthesis.

A simplified model system composed of a NADPH-dependent flavoprotein hydroxylase PgaE and a short-chain alcohol dehydrogenase/reductase (SDR) CabV was used to dissect a multistep angucycline modification redox cascade into several subreactions in vitro. We demonstrate that the two enzymes are sufficient for the conversion of angucycline substrate 2,3-dehydro-UWM6 to gaudimycin C. The flavoenzyme PgaE is shown to be responsible for two consecutive NADPH- and O(2)-dependent reactions, consistent with the enzyme-catalyzed incorporation of oxygen atoms at C-12 and C-12b in gaudimycin C. The two reactions do not significantly overlap, and the second catalytic cycle is initiated only after the original substrate 2,3-dehydro-UWM6 is nearly depleted. This allowed us to isolate the product of the first reaction at limiting NADPH concentrations and allowed the study of the qualitative and kinetic properties of the separated reactions. Dissection of the reaction cascade also allowed us to establish that the SDR reductase CabV catalyzes the final biosynthetic step, which is closely coupled to the second PgaE reaction. In the absence of CabV, the complete PgaE reaction leads invariably to product degradation, whereas in its presence, the reaction yields the final product, gaudimycin C. The result implies that the C-6 ketoreduction step catalyzed by CabV is required for stabilization of a reactive intermediate. The close relationship between PgaE and CabV would explain previous in vivo observations: why the absence of a reductase gene may result in the lack of C-12b-oxygenated species and, vice versa, why all C-12b-oxygenated angucyclines appear to have undergone reduction at position C-6.