Data_Sheet_1_Catalytic Performance of a Class III Old Yellow Enzyme and Its Cysteine Variants.docx

Class III old yellow enzymes (OYEs) contain a conserved cysteine in their active sites. To address the role of this cysteine in OYE-mediated asymmetric synthesis, we have studied the biocatalytic properties of OYERo2a from Rhodococcus opacus 1CP (WT) as well as its engineered variants C25A, C25S and C25G. OYERo2a in its redox resting state (oxidized form) is irreversibly inactivated by N-methylmaleimide. As anticipated, inactivation does not occur with the Cys variants. Steady-state kinetics with this maleimide substrate revealed that C25S and C25G doubled the turnover frequency (kcat) while showing increased KM values compared to WT, and that C25A performed more similar to WT. Applying the substrate 2-cyclohexen-1-one, the Cys variants were less active and less efficient than WT. OYERo2a and its Cys variants showed different activities with NADPH, the natural reductant. The variants did bind NADPH less well but kcat was significantly increased. The most efficient variant was C25G. Replacement of NADPH with the cost-effective synthetic cofactor 1-benzyl-1,4-dihydronicotinamide (BNAH) drastically changed the catalytic behavior. Again C25G was most active and showed a similar efficiency as WT. Biocatalysis experiments showed that OYERo2a, C25S, and C25G converted N-phenyl-2-methylmaleimide equally well (81–84%) with an enantiomeric excess (ee) of more than 99% for the R-product. With cyclic ketones, the highest conversion (89%) and ee (>99%) was observed for the reaction of WT with R-carvone. A remarkable poor conversion of cyclic ketones occurred with C25G. In summary, we established that the generation of a cysteine-free enzyme and cofactor optimization allows the development of more robust class III OYEs.</p

Data_Sheet_1_Pyridine Nucleotide Coenzyme Specificity of p-Hydroxybenzoate Hydroxylase and Related Flavoprotein Monooxygenases.pdf

p-Hydroxybenzoate hydroxylase (PHBH; EC 1.14.13.2) is a microbial group A flavoprotein monooxygenase that catalyzes the ortho-hydroxylation of 4-hydroxybenzoate to 3,4-dihydroxybenzoate with the stoichiometric consumption of NAD(P)H and oxygen. PHBH and related enzymes lack a canonical NAD(P)H-binding domain and the way they interact with the pyridine nucleotide coenzyme has remained a conundrum. Previously, we identified a surface exposed protein segment of PHBH from Pseudomonas fluorescens involved in NADPH binding. Here, we report the first amino acid sequences of NADH-preferring PHBHs and a phylogenetic analysis of putative PHBHs identified in currently available bacterial genomes. It was found that PHBHs group into three clades consisting of NADPH-specific, NAD(P)H-dependent and NADH-preferring enzymes. The latter proteins frequently occur in Actinobacteria. To validate the results, we produced several putative PHBHs in Escherichia coli and confirmed their predicted coenzyme preferences. Based on phylogeny, protein energy profiling and lifestyle of PHBH harboring bacteria we propose that the pyridine nucleotide coenzyme specificity of PHBH emerged through adaptive evolution and that the NADH-preferring enzymes are the older versions of PHBH. Structural comparison and distance tree analysis of group A flavoprotein monooxygenases indicated that a similar protein segment as being responsible for the pyridine nucleotide coenzyme specificity of PHBH is involved in determining the pyridine nucleotide coenzyme specificity of the other group A members.</p

MonoQ anion exchange chromatography elution profiles of flavodoxin and apoflavodoxin.

<p>(A) Flavodoxin (40 μM). (B) Flavodoxin (40 μM), kept in the presence of 10 mM DTT for a period of 10 min. (C) Apoflavodoxin (50 μM). (D) Apoflavodoxin (50 μM), kept in the presence of 10 mM DTT for a period of 10 min. Gradient composition: buffer A is 25 mM Tris-HCl pH 8.0, and buffer B is 25 mM Tris-HCl pH 8.0, containing 1 M KCl. Flow rate is 1.0 mL/min. Dashed lines show conductivities of elution buffers. The molecule eluting at 7 mL is DTT. Temperature is 25°C.</p

Oxidation states of protein cysteines, and their reversibility by DTT.

<p>Schematic diagram showing steps involved in hydrogen peroxide-induced oxidation and DTT-induced reduction of protein cysteines.</p

Cartoon drawing of the X-ray structure of flavodoxin from <i>A. vinelandii</i>.

<p>α-Helices are shown in red, β-strands in blue and loops in white. The FMN cofactor is coloured yellow and the backbone of residue 69 is coloured magenta. Note that this residue is in immediate vicinity of FMN. The X-ray structure is of the C69A variant of the protein (pdb ID 1YOB) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Alagaratnam1" target="_blank">[30]</a>, in which the single cysteine at position 69 is replaced by alanine. This protein variant is largely similar to flavodoxin regarding both redox potential of holoprotein and stability of apoprotein <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Steensma1" target="_blank">[42]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-vanMierlo2" target="_blank">[54]</a>.</p

Monitoring of (apo)flavodoxin under H₂O₂-induced oxidative stress by LC-MS.

<p>For clarity, the zoomed in LC-MS regions that display the +10 charge state of monomer and the +19 charge state of dimer are shown (i.e., m/z range of 1940 to 2070); however, analysis is done on the whole m/z range. (A) Apoflavodoxin. (B) Apoflavodoxin incubated for 30 min with 10 mM H₂O₂. (C) Apoflavodoxin incubated for 30 min with 100 mM H₂O₂. (D) Apoflavodoxin incubated for 30 min with 200 µM NBD-Cl and 100 mM H₂O₂. (E) Flavodoxin incubated for 30 min with 190 µM NBD-Cl and 100 mM H₂O₂. Protein concentration is 5 µM and incubations were done at room temperature. M represents apoflavodoxin monomer with non-oxidised thiol; MO, MO2 and MO3 are the sulfenic, sulfinic and sulfonic acid states of apoflavodoxin, respectively. M-NBD is monomer protein with the thiol adduct of NBD-Cl, MO-NBD is monomer protein with the sulfenic acid adduct of NBD-Cl, and D represents disulfide-linked apoflavodoxin dimer.</p

LC-MS and native MS spectra of flavodoxin.

<p>(A) LC-MS spectrum of flavodoxin. (B) Nano-electrospray mass spectrum of flavodoxin. M_H⁺ⁿ represents flavodoxin monomer with n positive charges, and M⁺ⁿ is apoflavodoxin monomer with n positive charges. (C and D) Spectra of area indicated by the grey contour in (A) and (B), respectively. Flavodoxin (5 µM) is in 50 mM ammonium acetate, 0.1 mM EDTA, pH 6.8.</p

Spectroscopic characteristics of apoflavodoxin modified with NBD.

<p>Apoprotein (20 μM) before (solid line) and after (dashed line) incubation with 200 μM NBD-Cl for 1 hour. Modified protein absorbs maximally at 420 nm, which is characteristic for the presence of a thiol-NBD conjugate <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0041363#pone.0041363-Ellis1" target="_blank">[47]</a>.</p

LC-MS analysis of (apo)flavodoxin.

<p>Monomeric protein species are labelled M. Molecular masses of NBD-Cl and NBD are 199.6 and 164.1 Da, respectively. Expected mass and measured mass are average masses.</p