Draw The Most Likely Product Of The Reaction Between Levatrol And Flavin Monooxygenases?
Org Biomol Chem. Author manuscript; available in PMC 2020 Feb vi.
Published in final edited class as:
PMCID: PMC6365201
NIHMSID: NIHMS1008495
Characterization of the Flavin Monooxygenase Involved in Biosynthesis of the Antimalarial FR-900098
Kim Nguyen
aneDepartment of Biochemistry, University of Illinois at Urbana-Champaign, 600 Southward Mathews Avenue, Urbana, IL 61801, USA
Matthew A. DeSieno
2Department of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, Usa
vInstitute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, United states
Brian Bae
iDepartment of Biochemistry, University of Illinois at Urbana-Champaign, 600 Southward Mathews Artery, Urbana, IL 61801, Us
5Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, U.s.a.
Tyler W. Johannes
2Department of Chemical and Biomolecular Engineering, Academy of Illinois at Urbana-Champaign, 600 Southward Mathews Avenue, Urbana, IL 61801, USA
Ryan E. Cobb
2Section of Chemical and Biomolecular Engineering, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
5Constitute for Genomic Biology, Academy of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
Huimin Zhao
1Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, United states
2Section of Chemic and Biomolecular Technology, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
3Department of Chemical science, University of Illinois at Urbana-Champaign, 600 Southward Mathews Artery, Urbana, IL 61801, U.s.a.
4Heart for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 Due south Mathews Artery, Urbana, IL 61801, USA
5Constitute for Genomic Biology, University of Illinois at Urbana-Champaign, 600 S Mathews Avenue, Urbana, IL 61801, USA
Satish Yard. Nair
1Department of Biochemistry, University of Illinois at Urbana-Champaign, 600 Southward Mathews Avenue, Urbana, IL 61801, USA
iiiSection of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, United states
4Heart for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, 600 Southward Mathews Avenue, Urbana, IL 61801, USA
5Institute for Genomic Biological science, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA
Abstract
The latter steps in this biosynthetic pathway for the antimalarial phosphonic acid FR-900098 include the installation of a hydroxamate onto three-aminopropylphosphonate, which is catalyzed by the consecutive actions of an acetyltransferase and an amine hydroxylase. Here, we present the 1.6 Å resolution co-crystal construction and accompanying biochemical characterization of FrbG, which catalyzes the hydroxylation of aminopropylphosphonate. We show that FrbG is a flavin-dependent N-hydroxylating monooxygenase (NMO), which shares a like overall construction with flavin-containing monooxygenases (FMOs). Notably, we as well show that the cytidine-5'-monophosphate moiety of the substrate is a critical determinant of specificity, distinguishing FrbG from other FMOs in that the nucleotide cofactor-bounden domain also serves in conferring substrate recognition. In the FrbG-FAD+-NADPH co-crystal structure, the C4 of the NADPH nicotinamide is situated near the N5 of the FAD isoalloxazine, and is oriented with a distance and stereochemistry to facilitate hydride transfer.
Keywords: Flavin-containing monooxygenases, phosphonic acids, catalytic mechanism
Graphical Abstract
Introduction
The rapid admission to genomic information has spurred a renewed involvement in the biosynthesis of natural products that contain phosphonate or phosphinate groups. Phosphonates are characterized by the presence of a hydrolytically inert C-P bond, and the biological activities of such compounds derive from their structural similarity to the corresponding labile phosphate ester. Studies by Seto and co-workers established that the thermodynamically unfavorable C-P bond germination reaction is catalyzed by the enzyme phosphoenolpyruvate mutase, and subsequent characterizations of phosphonate biosynthetic enzymes take revealed several unusual catalysts. Contempo studies have resulted in the label of the biosynthetic clusters for several phosphonates, including the antibiotic fosfomycin, the herbicide phosphinothricin, the antifungal rhizocticin and the antimalarial candidate FR-900098 (chemical compound 1 in Figure 1a) [one].
(a) Chemical structures of (i) {"type":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098, (2) iii-aminopropylphosphonate, (iii) N-acetyl-3-aminopropylphosphonate, (four) CMP-5'-3-aminopropylphosphonate, and (5) CMP-v'-N-acetyl-iii-aminopropylphosphonate. Only compound 3 (CMP-five'-3-aminopropylphosphonate) is a substrate for FrbG. (b) Absorbance spectra of FrbG. Trace A represents the fully oxidized course of the enzyme (at a concentration of 1 μM) in the absence of NADPH. Trace B was obtained immediately after addition of equimolar NADPH under aerobic conditions. Traces C-F were recorded i min, 5 min, 10 min and fifteen min after addition of the cofactor. The traces where the flavin is becoming more reduced are solid and where it is returning to the oxidized state at afterwards times are dashed. These demonstrate the tedious bounden of NADPH past the enzyme and formation of the C4α-hydroperoxyflavin intermediate.
FrbG was identified during the cloning and sequencing of the biosynthetic gene cluster for FR-900098 from Streptomyces rubellomurinus [two]. Initial sequence assay of FrbG using bioinformatics tools failed to place homologs in the poly peptide database with significant sequence similarity. Further characterization of the enzymes, including whole prison cell feeding studies, established that FrbG functions equally a N-hydroxylase during the biosynthesis of the hydroxamate moiety in FR-900098 [3]. Upon purification, in the absenteeism of exogenous compounds, the protein solution appears vivid yellow, and a UV-vis spectroscopic analysis of the purified protein in solution produces an absorbance spectrum characteristic of a flavin-containing molecule (Figure 1b), suggesting that FrbG is a flavoprotein monooxygenase.
Flavoprotein monooxygenases catalyze the regioselective monooxygenation on a wide variety of substrates including organic compounds, drugs, and xenobiotics. The flavin-containing monooxygenases (FMOs) and closely related microbial N-hydroxylating monooxygenases (NMOs), stand for a bracket of these enzymes in which a single polypeptide can catalyze both monooxygenation and flavin reduction using an NADPH cofactor as an electron donor to reduce the FAD. Dissimilar other monooxygenases, FMOs and NMOs do not crave the presence of the substrate for the reduction of the FAD prosthetic group by NADPH [4]. Instead, the enzyme-bound cofactor reacts with molecular oxygen to course an unstable C4α-hydroperoxyflavin intermediate, which is now primed to function as either an oxygenase (FMO) or N-hydroxylase (NMO) [5].
Prior biochemical and structural studies on FMOs suggests a model for catalysis in which the NADP(H) cofactor can participate in both flavin reduction and stabilization of the reactive C4α-hydroperoxyflavin against solvent-mediated decay to hydrogen peroxide [half dozen]. Crystal structures of eukaryotic and bacterial FMOs [6,7], along with that of the related phenylacetone Baeyer-Villiger monoxygenase [8], reveal a conformation in which the NADP+ cofactor is spring away from the flavin, in an orientation that would forestall hydride transfer, and in a conformation that is proposed to stabilize the C4α-hydroperoxyflavin. Consequently, the catalytic cycle of FMOs/NMOs is proposed to occur through conformational changes in the enzyme, involving a "pre-hydride transfer" conformation that promotes FAD reduction by NADPH, and is followed by a structural rearrangement to yield a "mail service-hydride transfer" conformation wherein the NADP+ is now poised for stabilization of the intermediate.
We report here biochemical characterization that establishes FrbG as the flavin-dependent Northward-hydroxylating monooxygenase involved in FR-900098 biosynthesis. We too present the i.6 Å resolution co-crystal structure of FrbG-FAD-NADPH complex. The structure of FrbG corresponds to a "pre-hydride transfer" conformation of an FMO family member, with the C4 of the nicotinamide and the N5 of the isoalloxazine in an orientation that would facilitate reduction of FAD by NADPH. Comparisons to structures of FMOs in the "post-hydride transfer" conformation [six,7], in which the nicotinamide and flavin are distally situated, show structural reorganizations that likely occur following dioxygen reduction of the flavin. The structural information suggests a number of active site residues that may play a part in the catalysis, and site-specific mutagenesis at these residues is used to establish the role of these residues in the catalytic wheel. Notably, we demonstrate that conjugation of the substrate 3-aminopropylphosphonate to cytidine-5'-monophosphate (CMP) is essential for catalysis, suggesting that the nicotinamide-binding domain may besides role in conferring substrate specificity.
Results and Discussion
Biochemical Assay.
Recombinant FrbG was heterologously produced in E. coli with a (cleavable) N-terminal polyhistidine fusion tag. Purification of protein through multiple chromatographic steps, specifically metal affinity, cation exchange, and size exclusion, yielded sample that was yellow in colour indicative of a bound cofactor. Steady state kinetic analysis was performed at a fixed concentration of NADPH with a series of putative substrates selected on the basis of the proposed biosynthetic pathway of FR-900098. Based on the originally proposed pathway, FrbG was believed to N-hydroxylate either iii-aminopropylphosphonate (3APn; chemical compound 2 in Figure 1a) or Northward-acetyl-iii-aminopropylphosphonate (Ac3APn; chemical compound 3 in Figure 1a) [ii]. Nevertheless, we take recently demonstrated that the afterwards steps in the pathway comprise phosphonate intermediates that are conjugated to CMP [three], and therefore, we as well analyzed the CMP conjugates of 3APn and Ac3APn (compounds 4 and 5, respectively, in Figure 1a). FrbG demonstrated activeness towards only i of these iv substrates, CMP-5'-3APn (four) (Table 1 and Figure S1 in Supporting Information).
Table 1.
Kinetic parameters of FrbG
| Substrate | k cat (min−1) (mean ± SD) | KM (μM) (hateful ± SD) | k true cat / One thousandThousand (mM−1 min−i) |
|---|---|---|---|
| CMP-v'-3APn (4) | five.ix ± 0.4 | fifteen.1 ± one.5 | 391 |
| CMP-five'-Ac3APn (5) | NR | ||
| 3APn (2) | NR | ||
| Ac3APn (3) | NR |
Fully oxidized FrbG displays the expected UV absorption maxima at 265, 369 and 450 nm with a shoulder at 475 nm (Effigy 1b, trace A). Stabilization of the C4α-hydroperoxyflavin intermediate has been well documented for several other FMOs [6,nine,10,11]. In these enzymes, mixing equimolar oxidized protein and NADPH in an aerated buffer solution lead to firsthand formation of the highly stable intermediate, with a characteristic elevation at ~360 nm [nine,12,13,14]. Upon addition of NADPH to FrbG, an increase in absorbance and shift to 349 nm was recorded, instead of the expected elevation (trace B). After 15 minutes, the spectrum resembled the C4α-hydroperoxyflavin intermediate (trace East). Also, the fully reduced form of FrbG could not exist obtained, fifty-fifty in the presence of 10-fold excess of NADPH. In typical FMOs, the enzyme volition get immediately reduced (seen as a shoulder instead of a peak at 450 nm). Due to the inherent NADPH oxidase action of these enzymes, the spectrum will slowly revert to the spectrum of the oxidized flavin every bit hydrogen peroxide (H2O2) is released from the FMO [6]. In the case of FrbG, in that location is a ho-hum decrease in the observed peak at 450 nm, instead of immediate formation of the fully reduced flavin (traces B-D). This peak reaches a minimum after approximately five minutes, followed by an increase in absorbance every bit the spectrum re-acquires the feature of oxidized flavin (traces E-F). This occurrence can be explained past the proposed "gatekeeper" role of NADP+ in FMOs [15]. The normally tightly leap cofactor helps stabilize the C4α-hydroperoxyflavin intermediate, preventing rapid formation of HtwoO2 in the absence of substrate. The fully reduced flavin was unable to be obtained under anaerobic conditions equally well with the addition of NADP+. The inclusion of the oxidized cofactor inhibited the speed of flavin reduction compared to NADPH alone. These studies suggest alternating binding betwixt the substrate and the cofactor, making the C4α-hydroperoxyflavin intermediate more susceptible to NADPH oxidase action compared to the case where NADP+ is the final product to exist released.
FrbG has relatively lower activity compared to other FMOs (thou true cat /K M of ~ x4 Thou−ane southward−1 as compared to tenv to tensix M−ane southward−1 for other characterized FMOs), which is probable the outcome of several factors. Enzymes in secondary metabolism tend to accept lower rates compared to those within master metabolism, so as to not divert key metabolites and resources away from critical reactions. This phenomenon is likely seen here with the susceptibility of FrbG to NADPH oxidase activity. Finally, the FrbG crystal construction, which will be discussed further, independent NADPH rather than the oxidized NADP+. Equally a event, the equilibrium between the two must favor NADPH that would also lower the overall charge per unit of the enzyme reaction.
Overall Construction.
In order to gain molecular insights into the mechanism of catalysis, we solved the construction of full-length FrbG to a resolution of 1.vi Å (Figure 2a). Despite the fact that no exogenous ligands were added to the crystallization buffers, the crystals had a very yellowish advent, and clear electron density can be observed for both NADPH and FAD in both experimental electron density maps and deviation maps calculated with phases from the last refined model (Figure 3a). The non-planar electron density observed for the nicotinamide band is consistent with the presence of the reduced class of the cofactor. In the crystal, there is one poly peptide-NADPH-FAD complex per disproportionate unit, consistent with the monomeric state observed for the enzyme in solution (Figure S2). The construction has been refined to a terminal free R factor of 22.1% (see Tabular array 2 for relevant crystallographic statistics).
(a) Ribbon diagram derived from the 1.six Å resolution structure of FrbG with the NADP+ binding domain colored in cyan and the FAD bounden domain colored in magenta. The coordinates for each of the ligands are shown as stick figures. (b) Structure-based sequence alignment of FrbG with other flavin dependent monooxygenases. Secondary structure elements demarcated in the alignment correspond to those noted in the ribbon diagram.
(a) Electron density maps calculated using Fourier coefficients Fobs – Fcalc with phases derived from the refined i.6 Å resolution structure of FrbG calculated with the coordinates for NADP+ and FAD omitted prior to 1 round of crystallographic refinement. The map is contoured at 3σ (blue mesh) and 8σ (red mesh) and the final refined coordinates for each ligand are superimposed. (b) Poly peptide residues involved in the binding of the NADP+ are shown in yellow, the NADP+ cofactor is shown in orange and the isoalloxazine ring of FAD is shown in majestic. (c) Protein residues (yellow) involved in interactions with FAD (purple). Solvent molecules within the first hydration shell are shown every bit cyan spheres.
Table two.
Data collection, phasing and refinement statistics
| Hi-resolution | SeMet FrbG | |
|---|---|---|
| Data collection | ||
| Space group | C2 | C2 |
| Unit cell dimensions | ||
| a, b, c (Å) | 121.3, 64.seven, 69.2 | 121.3, 64.6, 69.i |
| α, β, γ (°) | xc.0, 124.7, ninety.0 | xc.0, 124.8, 90.0 |
| Resolution (Å) | 50–1.six (1.66–one.6) 1 | fifty–1.viii (1.86–one.viii) ane |
| Rsym (%) 2 | 5.8 (27.i) | 6.ix (xx.half-dozen) |
| I / σ(I) | 42.ane (4.8) | 37.six (8.five) |
| Completeness (%) | 97.2 (88.four) | 97.7 (96.3) |
| Back-up | 7.five (v.ix) | v.9 (5.vi) |
| |FH|/e (anomalous) | 1.27 | |
| FOM/DM FOM iii | 0.376/0.76 | |
| Refinement | ||
| Resolution (Å) | 25.0–1.6 | 50–1.8 |
| Number of reflections | 53,510 | 37,762 |
| Rwork / Rfree 4 | 19.3%/22.one% | xviii.seven%/21.vii% |
| Number of atoms | ||
| Protein | 3122 | 3156 |
| Solvent | 487 | 568 |
| NADP | 48 | 48 |
| FAD | 53 | 53 |
| Average B value | ||
| Protein | 19.eight | 20.8 |
| Solvent | 31.9 | 35.3 |
| NADP | 15.ix | xvi.4 |
| FAD | 11.8 | xi.5 |
| R.m.s deviations | ||
| Bond lengths (Å) | i.64 | ane.35 |
| Bond angles (°) | 0.006 | 0.007 |
FrbG is composed of two structural domains (Figure 2a,b). Residues 167 – 340 grade a small (NADPH binding) domain and residues 1 – 166 along with 341 – 430 course a larger single (FAD bounden) domain. Each of the 2 motifs contains a β-α-β motif characteristic of mononucleotide binding sequences. The two domains are situated shut to i another such that a single channel that extends from one domain to the other is nestled within the core of the poly peptide. The region where the prosthetic group, FAD, and NADPH interact is located at the interface of the larger and smaller domains. The NADPH cofactor is sandwiched between the re side of the flavin and the carboxy edge of the fundamental parallel β-sheet and is enveloped predominantly past the smaller domain of FrbG.
A DALI structure-based comparison against the Protein Data Bank using the intact structure of FrbG identifies very few structural homologs, and is likely the consequence of various insertions and structural rearrangements between the two domains that distinguishes FrbG from other FMOs. Bioinformatics analysis using each of the isolated domains identifies the FAD-bounden domain of the thioredoxin reductase TrxR from Helicobacter pylori [16] as a structural homolog of the larger FAD-bounden domain (RMS deviation of 3.3 Å over 271 Cα atoms with thirteen% sequence identity) and the NADPH-binding domains of the FMO from Schizosaccharomyces pombe (RMS deviation of 3.two Å over 287 Cα atoms with 18% sequence identity) and Methylophaga sp. SK1 (RMS divergence of 3.3 Å over 285 Cα atoms with 14% sequence identity). Although the overall fold of FrbG is similar to those of other FMOs, the enzyme is distinguished by insertions of several secondary structural elements (vide infra and Figure S3).
FAD Binding Domain.
The nucleotide-binding motif GGGPAG forms the loop connecting the carboxy end of strand β1 to helix α1 and stabilizes binding of the adenosine diphosphate portion of FAD (Figure 3b). The glycine-rich region of the motif positions the central office of FAD close to the poly peptide framework. The amino end of helix α1, specifically the nitrogen of Ala-fourteen, makes a hydrogen bond with the pyrophosphate group O1P of FAD. The adenine nucleotide is stabilized by hydrogen bonding contacts to the main chain nitrogen atoms of Ala-41 and Val-131 and the carbonyl oxygen of Val-131. The prosthetic group is well solvated in the aqueduct with h2o molecules mediating poly peptide contacts. A well-localized h2o molecule farther anchors the flavin pyrophosphate group to the GGGPAG motif. This bound water molecule hydrogen bonds with the nitrogen atoms of Gly-12 and Gly-xv, the carbonyl oxygen of Gly-ten, and O1P of the flavin phosphate. An oxygen atom of the ribitol group along with an oxygen atom and several nitrogen atoms from the isoalloxazine ring of the flavin nucleotide interact with the big domain via h2o molecules. In the enzyme-FAD-NADPH complex, the isoalloxazine band of FAD is stacked with the nicotinamide of NADP+ (Effigy 3c). The oxygen atoms, O2 and O4, of the isoalloxazine band form hydrogen bonds with the main chain nitrogens of Val-412 and Asn-58, respectively. Although the flavin nucleotide extends toward the interaction domain to engage in stacking interactions with the nicotinamide, the prosthetic group remains predominantly enclosed in the larger domain of FrbG surrounded by a network of water molecules.
Structural and biochemical studies of FMO from S. pombe identified an Asn-91 residue located near the isoalloxazine ring of FAD that is believed to be directly involved in the catalytic mechanism [7]. This asparagine is thought to be crucial in supplying molecular oxygen to the isoalloxazine ring and stabilizing the resultant C4α-hydroperoxyflavin intermediate. FrbG has Asn-58 located at an coordinating position most the isoalloxazine ring and the Nδ2 of Asn-58 is 2.98 Å from a water molecule. This same water molecule is 3.fourscore and 2.96 Å from C4α and N5 of FAD, respectively. Initial refinement of S. pombe FMO had identified a water molecule in a comparable location that was later modeled with a dioxygen molecule [seven]. Dioxygen molecules near enzyme active sites take been observed with naphthalene dioxygenase and cytochromes P450 likewise [17,eighteen]. Mutation of Asn-58 to Ala in FrbG demonstrated its importance in the catalytic bike of the enzyme. The FrbG-N58A variants exhibited robust NADPH oxidase action, but activity with the CMP-5'-3APn substrate was compromised relative to the wild-blazon (Effigy S4). These data advise that NADPP oxidation is uncoupled to substrate hydroxylation in this variant. I plausible explanation for this observation is that FrbG-N58A may not exist able to sufficiently stabilize the C4α-hydroperoxyflavin intermediate to comport out the hydroxylation of the substrate.
NADP(H) Binding Domain.
The NADPH cofactor binds on the re side of the flavin and is situated closely to the 2nd nucleotide-bounden motif, GGAHS (Figure 3c). The nitrogen atoms of Gly-219 hydrogen bond to the iii'-OH of ribose of the adenine group and the primary concatenation nitrogen atoms of His-221 and Ser-222 anchor the nicotinamide pyrophosphates via hydrogen bonding. This glycine-rich region, which shares structural homology to the FAD nucleotide-binding motif, forms a brusk loop connecting the carboxy terminate of strand β5 to helix α5. A side chain nitrogen of residue Arg-250 hydrogen bonds to the phosphate present on the 2' carbon of the adenosine ribose of NADPH. The remaining side chain nitrogens of Arg-250 appoint in stacking interactions with the adenine nucleotide of NADPH. In improver, numerous water molecules are bound within the channel and make hydrogen-bonding contacts with the protein as well as the cofactor. Water molecules, along with the side chain oxygen of Asp-410, stabilize the nicotinamide ribose equally the reduced nicotinamide extends toward the interaction domain to stack with the isoalloxazine band of the flavin moiety of FAD.
NADPH is bound at the carboxy edge of the primal parallel β-canvass and is enveloped predominantly by the smaller domain of FrbG. Binding of NADPH in the smaller domain buries 101 Å2 of surface area, in contrast with the 237 Å2 of surface surface area buried upon FAD binding. Consequently, NADP+ is not every bit strongly embedded within FrbG as FAD since both the adenine nucleotide and pyrophosphates of NADPH are exposed to the solvent, whereas FAD'due south adenine group is deeply embedded within FrbG. Given the volume occupied past the ligands, it is likely that following the reduction of the flavin, NADP+ must be displaced from the active site prior to binding of the CMP-5'-3APn substrate, consequent with biochemical observations on other flavin monooxygenases.
Given our experimental data that only the CMP conjugate of the phosphonate (i.east. 4) is a viable substrate for FrbG, the NADP(H) binding domain probable also serves in substrate selectivity. Specifically, the CMP-5'-3APn (four) substrate may exist recognized by virtue of the nucleotide group of the cohabit. A docking model of CMP-5'-3APn (4) bound to the NADP(H) binding-domain, based on the superposition of the nucleobases of the substrate with cofactor NADPH provides a rationale for this specificity (Figure S5). The model suggests that binding of the CMP at a position like to that of NADPH results in placement of the amine of 3APn directly in a higher place the isoalloxazine ring, adjacent to C4α where molecular oxygen is suggested to bind. The orientation of the 3APn amine is further stock-still by His-221. FrbG-H221A had significantly higher rates for both NADPH oxidation and substrate hydroxylation compared to the wild-blazon FrbG. The alanine exchange at this residuum is consistent with a part for His-221 in the reaction machinery (Figure S4).
Machinery of FrbG Activity.
Every bit a major electron carrier in oxidation-reduction reactions, the isoalloxazine ring of FAD is first reduced to FADH2 via a hydride transfer from C4 of NADPH to the N5 atom of the flavin. The reduced flavin afterwards reacts with molecular oxygen to class a C4α-hydroperoxyflavin intermediate, FAD-OOH [19]. This peroxyflavin intermediate has been shown to be stable for minutes to hours at 4 °C and has been spectroscopically observed [20,21,22,23]. In one case a substrate with a nucleophilic atom is in shut proximity to the peroxyflavin intermediate, the nucleophilic attack conducted by the substrate results in the oxygenation or hydroxylation of the substrate by 1 cantlet of molecular oxygen, whereas the other cantlet is released equally h2o during the reaction [24].
Prior studies of FMOs from Due south. pombe and Methylophaga sp. SK1 show that the flavin N5 and the nicotinamide C4 are at an orientation and distance incompatible with hydride transfer (inter-diminutive altitude of 5.3 Å) (Figure 4c). This "post hydride transfer" conformation has been suggested to facilitate the stabilization of the C4α-hydroperoxyflavin past shielding the active site and providing a suitable hydrogen-bonding surroundings for the intermediate. Our FrbG-FAD-NADPH complex construction reveals close stacking of the nicotinamide ring with the isoalloxazine ring, with C4 of the nicotinamide 3.74 Å from N5 of the isoalloxazine band (Figure 4a,b). Attempts to soak crystals of FrbG with NADP+ resulted in immediate slap-up, consequent with the prevalent hypothesis that similar enzymes undergo a structural rearrangement following the flavin reduction by NADPH, to form the inactive conformation that has previously been observed crystallographically.
A comparison of the active sites in FrbG and S. pombe FMO well-nigh the vicinity of the NADP(H) and FAD. (a,c) The active site of FrbG with poly peptide residues shown in xanthous, the FAD shown colored in pinkish and the NADPH shown colored in tan. The nicotinamide C4 is located most the N5 of the isoalloxazine at a distance and orientation that would facilitate hydride transfer. A number of secondary structure elements (shown in pinkish) support the position of the NADPH near the flavin. (b,d) In dissimilarity, within the active site of S. pombe FMO, the nicotinamide (in blueish) is situated too far away from the FAD (in dark-green) for productive hydride transfer. The secondary structural elements (in pink) that could orient the NADP+ are situated abroad from the active site. A comparing of the 2 structures may perhaps reverberate the necessary conformational change that occurs during the catalytic bike.
Domain movements take been invoked as necessary for the catalytic cycles of FMOs and structurally related proteins, such as phenylacetone monooxygenase [eight]. Like domain movements have been observed in crystallographic studies of the topologically related thioredoxin reductase, in which the NADPH and FAD binding domains rotate by 67° in order to accomplish a conformation that is compatible with hydride transfer [25]. A comparison of the structures of FrbG ("pre hydride transfer") with those of FMOs from S. pombe and Methylophaga sp. SK1 ("post hydride transfer") suggests some of the structural changes that may accompany the flavin reduction (Figure 4d,eastward). The topology of FrbG is characterized by three helical insertions (α14-α16) between the 4th and fifth β-strand of the smaller nucleotide-binding domain. These helices are situated at similar locations as seen for the two carboxy-terminal helices in the structures of other FMOs, and are suggested to undergo a conformational movement in response to hydride transfer. The loftier thermal (B) factors observed for the Methylophaga sp. SK1 FMO in this region are idea to bespeak conformational flexibility that may facilitate structural reorganization [six]. Based on our structural comparison, the relative orientations of these helices are representative of the reorganization that accompanies hydride transfer. In the FrbG construction, helix α13 is situated near the NADPH in society to orient His-221 for stabilization of the nicotinamide. The analogous helix in both from Schizosaccharomyces pombe and Methylophaga sp. SK1 FMOs orient away from the nucleotide [6,7], and this shift may reflect another reorganization that occurs following hydride transfer.
The biochemical analysis and crystal construction described here led us to advise a reaction machinery for the North-hydroxylation catalyzed past FrbG (Figure five). Similar to known N-hydroxlases, the cofactor NADPH first enters the enzyme and reduces FAD to course FADH2. In the absenteeism of substrate, the C4α-hydroperoxyflavin intermediate can typically at present be formed by the addition of molecular oxygen. Alternatively, the UV-vis spectroscopic analysis indicates slow formation of the intermediate and susceptibility towards NADPH oxidase activity, which may be consistent with formation of the C4α-hydroperoxyflavin intermediate. Our crystallographic structure of FrbG-NADPH-FAD circuitous demonstrates the being of this stable "pre hydride transfer" intermediate, which is also consistent with our inability to obtain a fully reduced FrbG.
Proposed catalytic mechanism of FrbG. As drawn, acceptance of molecular oxygen to form the C4α-hydroperoxyflavin intermediate occurs prior to binding of the substrate. Yet, it is too formally possible that C4α-hydroperoxyflavin germination occurs after substrate is leap.
The lack of a substrate-binding pocket within the active site in the FrbG-NADPH-FAD complex suggests that substrate and cofactor occupy overlapping binding sites, consistent with observations in the Southward. pombe FMO, where the substrate methimazole occupies the same binding site as NADP+ [7]. Inhibition of the initial velocity was monitored at increasing concentrations of the substrate (Effigy S6). These data suggest that the CMP-five'-3APn (4) substrate and NADPH cofactor compete for the same bounden pocket. Prior structural studies on S. pombe FMO also evidence that the enzyme remains in the "post hydride transfer" conformation as the NADP+ is displaced from the binding site, suggesting that cofactor displacement must be fast enough that the C4α-hydroperoxyflavin does non decay. Likewise, in FrbG, following hydride ion transfer to the flavin, NADP+ is displaced from the active site in lodge to adjust bounden of the CMP conjugated 3-aminopropylphosphonate substrate, with the C4α-hydroperoxyflavin so rapidly transferring jump molecular oxygen onto the substrate. Following the N-hydroxylation of the substrate, a water molecule is released and FADHtwo is oxidized to FAD in preparation for some other catalytic cycle. CMP recognition by the NADPH/substrate binding site of FrbG contributes to tight substrate specificity in that the substrate would be of the appropriate length to access the C4α-hydroperoxyflavin intermediate inside the aqueduct for catalysis to occur.
Following FrbG-catalyzed hydroxylation, a chemical compound is observed by LC-MS with the correct m/z to be the nitroso derivative of the hydroxylamine production. This chemical compound could arise via a second FrbG-catalyzed hydroxylation cycle to the dihydroxy-intermediate, followed by a spontaneous dehydration to the nitroso. Even so, rapid turnover of the hydroxylamine product to the nitroso derivative ensued even following removal of FrbG, and was further observed for the chemically synthesized hydroxylamine post-obit exposure to air (Figure S7). As a result, this second oxidation step is most likely non-enzymatic, every bit has been observed for other hydroxylamine-containing compounds [26].
Conclusion
FrbG is a novel flavin dependent monooxygenase that catalyzes the synthesis of the CMP-Northward-hydroxy-three-aminopropylphosphonate intermediate during {"blazon":"entrez-nucleotide","attrs":{"text":"FR900098","term_id":"525219861","term_text":"FR900098"}}FR900098 biosynthesis. The crystal structure of FrbG-NADPH-FAD complex reveals a 2 domain architecture with each domain harboring nucleotide binding motifs to anchor the prosthetic group (FAD) and stabilize the cofactor (NADPH) required for catalysis. Prior studies of other FMOs in the "post hydride transfer" conformation propose a "moonlighting" role for the cofactor, where NADPH promotes FAD reduction and then stabilizes the C4α-hydroperoxyflavin intermediate. A comparison of these structures with that of FrbG reveals structural reorganizations that probable occur post-obit flavin reduction. Our current studies add to what little is known about the biosynthesis of phosphonates and help in our efforts of increasing the efficiency, improving the efficacy, and enhancing the production of FR-900098, an antibiotic that has been clinically proven to be effective confronting malaria. This work has the potential of expanding the therapeutic applications of FR-900098 and its derivatives beyond parasitic and bacterial infections.
Materials and Methods
Cloning, expression and purification.
Full-length FrbG was cloned into the pET28a(+) vector and transformed into E. coli BL21(DE3) competent cells. Transformed cells were cultured overnight at 37 °C in Luria broth medium (BD Biosciences, San Jose, CA) with the appropriate antibiotic (50 μg/ml of kanamycin). The cultures were diluted ane:100 with the same medium and grown to an OD 600 ~ 0.8. Protein overexpression was induced with a final concentration of 1 mM isopropyl-β-D-thiogalactoside and cultures were incubated at 18 °C with agitation for eighteen hours. After centrifugation at 4000 rpm for thirty minutes the supernatant was discarded and the pellet was resuspended in 10 mM PBS, pH 8.0. Samples were lysed via the freeze/thaw and French Printing (3000–4000 psi) methods and centrifuged at 15000 rpm for i hour. The soluble fraction was collected and subjected to affinity chromatography with buffer containing twenty mM Tris, pH 8.0 and 500 mM NaCl in the absenteeism and presence of 250 mM imidazole, pH 8.0. SDS-Folio gel electrophoresis was used to appraise the presence of FrbG in the nerveless fractions. Cleavage and removal of the 6xHis-tag from FrbG was performed by the improver of thrombin (1 unit/ml) and CaCl2 (i mM) as the collected fractions dialyzed into 20 mM Tris, pH 8.0 and 100 mM KCl. The nerveless fractions were further purified by cation substitution and size exclusion chromatography with buffer substitution into 20 mM HEPES, pH 7.5 and 100 mM KCl. Concentration using a 10-kDa molecular mass limit centrifugal filter device (Millipore, Billerica, MA) resulted in a poly peptide yield of ~45 mg/ml. Selenomethionine-labeled protein was produced and purified in a similar style with the exception of growth in minimal media and addition of amino acids and selenomethionine prior to induction.
LC-MS Analysis of the FrbG reaction.
After incubating purified FrbG with the substrate CMP-5'-3-aminopropylphopshonate (CMP-5'-3APn) and NADPH, conversion to CMP-5'-N-hydroxy-3-aminopropylphopshonate (CMP-five'-H3APn) was monitored by HPLC. Product formation was further confirmed by LC-MS, where the new tiptop displayed the expected fragmentation (one thousand/z 459→322) of the hydroxylamine.
UV/Vis absorbance spectrum analysis.
Using a Cary 300 Bio UV-vis spectrophotometer (Varian, Cary, NC), yellow, purified FrbG (20 μM) in solution was analyzed and compared with the standard spectra of the buffer, twenty mM HEPES, pH vii.v, and 100 mM KCl, within which FrbG was stored.
Crystallization.
Purified and concentrated (v–x mg/ml) total-length FrbG was crystallized using the hanging driblet vapor diffusion method. Crystals grew at 8 °C in 3-μl drops in 0.2 G KCl, 0.1 Thousand magnesium acetate tetrahydrate, 0.05 K sodium cacodylate trihydrate, pH half dozen.5 and 10% (w/five) polyethylene glycol 8000 against a reservoir of the aforementioned composition. FrbG crystals took ~14–xxx days to form. Selenomethionine incorporated FrbG crystals were more reproducible than native FrbG crystals in the aforementioned crystallization status.
Construction determination of enzyme-FAD-NADPH complex.
FrbG native and selenomethionine crystals were cryoprotected with 1-minute soaks at 4 °C in female parent liquor containing 30% glycerol and immediately flash frozen with liquid nitrogen. Selenomethionine-incorporated amplitudes were nerveless, under standard cryogenic conditions, at a high-flux insertion device synchrotron beam line (Argonne National Laboratories, sector 21D, F, G), to a limiting resolution of ane.vi Å and the corresponding data processed using HKL2000 [27]. As the anomalous signal from this data fix was small owing to the depression symmetry of the crystals, another 1.8 Å resolution information prepare was nerveless using the inverse-axle method for crystallographic phasing. Heavy-atom sites were located using HySS [28] and imported into Abrupt [29] for maximum likelihood refinement. An electron density map calculated from the resultant phases was of excellent quality, permitting automatic and manual plumbing fixtures of the unabridged polypeptide chain using XtalView [30]. Iterative cycles of model building followed by circular of refinement using REFMAC5 [31] farther improved the model. Water molecules were added when the free R factors fell beneath xxx% and cofactors and prosthetic groups were simply added during the very tardily stages of refinement. The stereochemistry of the models was routinely monitored throughout the course of refinement using PROCHECK [32]. Crystal parameters, information drove parameters and refinement statistics for each of the structures are summarized in Tabular array ii.
Sequence and structural comparisons.
A preliminary search for sequence and structural homology was performed using Basic Local Alignment Search Tool (BLAST) on the National Center for Biotechnology Data (NCBI) Spider web server [33] and the Research Collaboratory for Structural Bioinformatics (RCSB) Protein Data Banking company [34]. The DALI server [35] was used to observe poly peptide fold similarity between FrbG and other known structures.
Kinetic analysis.
Kinetic assays were performed using a Varian Cary 100 Bio UV-visible spectrophotometer (Varian) in triplicate at xxx °C. A 100 μL mixture containing ane μM FrbG and substrate (concentrations varied from 0–two mM) in fifty mM HEPES buffer, pH vii.v was incubated for 5 minutes. The reaction was initiated past improver of 200 μM NADPH and so monitored past the decrease in absorbance at 340 nm or by product formation on LC-MS. The C4α-hydroperoxyintermediate was generated past addition of vii μM NADPH to FrbG in aerated buffer solution (seven μM in 50 mM HEPES, pH 7.5) at 4 °C [6].
Mutational analysis.
A megaprimer PCR method was used to generate the N58A and H221A site specific mutants of FrbG. Briefly, primers containing the designed mutations were used in the first PCR to create the megaprimers. The megaprimers were then used in a 2nd PCR to obtain the total-length mutants. Expression and purification of the two mutants followed the protocol listed above. A modified protocol to the kinetic analysis was used. A 100 μL mixture containing 5 μM wild blazon or mutant FrbG and 500 μM CMP-5'-3APn (not included for NADPH oxidase activity) in 50 mM HEPES buffer was incubated for 5 minutes. The reaction was initiated past addition of 200 μM NADPH and and then monitored by the decrease in absorbance at 340 nm.
Analysis of non-enzymatic oxidation of the hydroxylamine production.
Loss of the hydroxylamine production via not-enzymatic oxidation to the nitroso derivative was observed by 2 methods. First, the FrbG enzyme was removed from reaction mixtures (as described above) via diafiltration in a 10 kDa cutoff filter. The resulting mixtures were then incubated at 30°C, with samples nerveless and analyzed by LC-MS for the presence of the hydroxylamine product. 2nd, the nitroso derivative was purified by HPLC fractionation and anaerobically reduced back to the hydroxylamine using SmI2 following the method of Kende and Mendoza [36]. The hydroxylamine product of this reaction was analyzed past LC-MS both earlier and afterwards 30 minutes of aerobic exposure.
Supplementary Material
ESI
Acknowledgements
Nosotros give thanks Joseph Brunzelle and Keith Brister for facilitating data collection at the Life Sciences Collaborative Access Team (Sector 21) at Argonne National Labs. This work was supported by a program grant from the National Institute of General Medical Sciences GM077596 (H.Z. and S.Yard.N). Yard.A.D. acknowledges support from the United states National Institutes of Health under Ruth 50. Kirschstein National Inquiry Award 5 T32 GM070421 from the National Institute of General Medical Sciences. The funding sources had no role in written report design, information collection and assay, decision to publish, or training of the manuscript.
References
ane. Metcalf WW, van der Donk WA (2009) Biosynthesis of phosphonic and phosphinic acid natural products. Annu Rev Biochem 78: 65–94. [PMC costless article] [PubMed] [Google Scholar]
2. Eliot AC, Griffin BM, Thomas PM, Johannes TW, Kelleher NL, et al. (2008) Cloning, expression, and biochemical characterization of Streptomyces rubellomurinus genes required for biosynthesis of antimalarial chemical compound FR900098. Chem Biol 15: 765–770. [PMC free article] [PubMed] [Google Scholar]
3. Johannes TW, DeSieno MA, Griffin BM, Thomas PM, Kelleher NL, et al. (2010) Deciphering the belatedly biosynthetic steps of antimalarial chemical compound FR-900098. Chem Biol 17: 57–64. [PMC free article] [PubMed] [Google Scholar]
4. Cashman JR (2005) Some distinctions betwixt flavin-containing and cytochrome P450 monooxygenases. Biochem Biophys Res Commun 338: 599–604. [PubMed] [Google Scholar]
5. Ziegler DM (1993) Recent studies on the structure and function of multisubstrate flavin-containing monooxygenases. Annu Rev Pharmacol Toxicol 33: 179–199. [PubMed] [Google Scholar]
six. Alfieri A, Malito E, Orru R, Fraaije MW, Mattevi A (2008) Revealing the moonlighting role of NADP in the structure of a flavin-containing monooxygenase. Proc Natl Acad Sci U Southward A 105: 6572–6577. [PMC free article] [PubMed] [Google Scholar]
7. Eswaramoorthy S, Bonanno JB, Burley SK, Swaminathan Due south (2006) Machinery of action of a flavin-containing monooxygenase. Proc Natl Acad Sci U S A 103: 9832–9837. [PMC gratis article] [PubMed] [Google Scholar]
eight. Malito E, Alfieri A, Fraaije MW, Mattevi A (2004) Crystal structure of a Baeyer-Villiger monooxygenase. Proc Natl Acad Sci U S A 101: 13157–13162. [PMC free article] [PubMed] [Google Scholar]
nine. Beaty NB, Ballou DP (1981) The oxidative half-reaction of liver microsomal FAD-containing monooxygenase. J Biol Chem 256: 4619–4625. [PubMed] [Google Scholar]
10. Poulsen LL, Ziegler DM (1979) The liver microsomal FAD-containing monooxygenase. Spectral characterization and kinetic studies. J Biol Chem 254: 6449–6455. [PubMed] [Google Scholar]
xi. Beaty NB, Ballou DP (1980) Transient kinetic study of liver microsomal FAD-containing monooxygenase. J Biol Chem 255: 3817–3819. [PubMed] [Google Scholar]
12. Kemal C, Bruice TC (1976) Unproblematic synthesis of a 4a-hydroperoxy adduct of a 1,v-dihydroflavine: preliminary studies of a model for bacterial luciferase. Proc Natl Acad Sci U S A 73: 995–999. [PMC gratuitous commodity] [PubMed] [Google Scholar]
13. Poulsen LL, Ziegler DM (1995) Multisubstrate flavin-containing monooxygenases: applications of mechanism to specificity. Chem Biol Collaborate 96: 57–73. [PubMed] [Google Scholar]
14. Suh JK, Poulsen LL, Ziegler DM, Robertus JD (1996) Molecular cloning and kinetic label of a flavin-containing monooxygenase from Saccharomyces cerevisiae. Arch Biochem Biophys 336: 268–274. [PubMed] [Google Scholar]
15. Cashman JR (1995) Structural and catalytic properties of the mammalian flavin-containing monooxygenase. Chem Res Toxicol 8: 166–181. [PubMed] [Google Scholar]
xvi. Gustafsson TN, Sandalova T, Lu J, Holmgren A, Schneider G (2007) High-resolution structures of oxidized and reduced thioredoxin reductase from Helicobacter pylori. Acta Crystallogr D Biol Crystallogr 63: 833–843. [PubMed] [Google Scholar]
17. Karlsson A, Parales JV, Parales RE, Gibson DT, Eklund H, et al. (2003) Crystal construction of naphthalene dioxygenase: side-on binding of dioxygen to atomic number 26. Science 299: 1039–1042. [PubMed] [Google Scholar]
18. Schlichting I, Berendzen J, Chu K, Stock AM, Maves SA, et al. (2000) The catalytic pathway of cytochrome p450cam at diminutive resolution. Science 287: 1615–1622. [PubMed] [Google Scholar]
19. Massey V (1994) Activation of molecular oxygen by flavins and flavoproteins. J Biol Chem 269: 22459–22462. [PubMed] [Google Scholar]
20. Beaty NB, Ballou DP (1980) Microsomes, Drug oxidation, and chemical carcinogenesis. New York: Bookish Press; 299–302 p. [Google Scholar]
21. Ziegler DM, Poulsen LL, Duffel MW (1980) Microsomes, Drug oxidation, and chemical carcinogenesis. New York: Academic Press; 637–645 p. [Google Scholar]
22. Williams DE, Ziegler DM, Nordin DJ, Hale SE, Masters BS (1984) Rabbit lung flavin-containing monooxygenase is immunochemically and catalytically distinct from the liver enzyme. Biochem Biophys Res Commun 125: 116–122. [PubMed] [Google Scholar]
23. Jones KC, Ballou DP (1986) Reactions of the 4a-hydroperoxide of liver microsomal flavin-containing monooxygenase with nucleophilic and electrophilic substrates. J Biol Chem 261: 2553–2559. [PubMed] [Google Scholar]
24. Krueger SK, Williams DE (2005) Mammalian flavin-containing monooxygenases: construction/function, genetic polymorphisms and office in drug metabolism. Pharmacol Ther 106: 357–387. [PMC costless article] [PubMed] [Google Scholar]
25. Lennon BW, Williams CH Jr., Ludwig ML (2000) Twists in catalysis: alternating conformations of Escherichia coli thioredoxin reductase. Science 289: 1190–1194. [PubMed] [Google Scholar]
26. Li N, Korboukh VK, Krebs C, Bollinger JM Jr. (2010) Four-electron oxidation of p-hydroxylaminobenzoate to p-nitrobenzoate by a peroxodiferric complex in AurF from Streptomyces thioluteus. Proc Natl Acad Sci U Due south A 107: 15722–15727. [PMC gratuitous article] [PubMed] [Google Scholar]
27. Otwinowski Z, Borek D, Majewski W, Small-scale West (2003) Multiparametric scaling of diffraction intensities. Acta Crystallogr A 59: 228–234. [PubMed] [Google Scholar]
28. Grosse-Kunstleve RW, Adams PD (2003) Substructure search procedures for macromolecular structures. Acta Crystallogr D Biol Crystallogr 59: 1966–1973. [PubMed] [Google Scholar]
29. Bricogne G, Vonrhein C, Flensburg C, Schiltz 1000, Paciorek W (2003) Generation, representation and menstruum of phase information in construction determination: recent developments in and around SHARP ii.0. Acta Crystallogr D Biol Crystallogr 59: 2023–2030. [PubMed] [Google Scholar]
30. McRee DE (1999) XtalView/Xfit--A versatile program for manipulating atomic coordinates and electron density. J Struct Biol 125: 156–165. [PubMed] [Google Scholar]
31. Murshudov GN, Vagin AA, Dodson EJ (1997) Refinement of macromolecular structures by the maximum-likelihood method. Acta Crystallogr D Biol Crystallogr 53: 240–255. [PubMed] [Google Scholar]
32. Laskowski RA, Rullmannn JA, MacArthur MW, Kaptein R, Thornton JM (1996) AQUA and PROCHECK-NMR: programs for checking the quality of protein structures solved by NMR. J Biomol NMR viii: 477–486. [PubMed] [Google Scholar]
33. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, et al. (1997) Gapped Boom and PSI-Nail: a new generation of protein database search programs. Nucleic Acids Res 25: 3389–3402. [PMC free article] [PubMed] [Google Scholar]
34. Berman HM, Battistuz T, Bhat TN, Bluhm WF, Bourne PE, et al. (2002) The Protein Data Bank. Acta Crystallogr D Biol Crystallogr 58: 899–907. [PubMed] [Google Scholar]
35. Holm L, Sander C (1993) Protein construction comparison by alignment of distance matrices. J Mol Biol 233: 123–138. [PubMed] [Google Scholar]
36. Kende Every bit, Mendoza JS (1991) Controlled reduction of nitroalkanes to alkyl hydroxylamines or amines by samarium diiodide. Tetrahedron Letters 32: 1699–1702. [Google Scholar]
Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365201/
Posted by: mondragontrionevered68.blogspot.com
0 Response to "Draw The Most Likely Product Of The Reaction Between Levatrol And Flavin Monooxygenases?"
Post a Comment