Fig 1.
Metabolic pathway for the conversion of lysine to adipic acid proposed by Burgard A. et al. [1].
The reaction number R05099 according to the Kyoto Encyclopedia of Genes and Genomes is given for the known reaction. For known enzymes, the E.C. numbers according to the Braunschweig Enzyme Database are given. akg = alpha-ketoglutaric acid, Glu-L = L-glutamic acid.
Table 1.
Plasmids used in this study.
Table 2.
E. coli strains used in this study.
Fig 2.
Metabolic pathways for the conversion of lysine to adipic acid.
Our alternative pathway. Different types of enzymatic reactions are shown in different colors: removal of NH2 from α-carbon (yellow), reduction of unsaturated α,β bond (pink), removal of the terminal NH2 (green), and oxidation of aldehyde to carboxylic acid (blue). The enzymatic reactions targeted in this study are encircled. The reaction numbers according to the Kyoto Encyclopedia of Genes and Genomes are given for known reactions. The E.C. numbers according to the Braunschweig Enzyme Database are given for known enzymes. akg = alpha-ketoglutaric acid, Glu-L = L- glutamic acid.
Fig 3.
Substrates used for enzymatic activity determination in this study.
*Obtained from scifinder.cas.org. For trans-2-hexenedioic acid the most acidic pKa is given. ** Positive control.
Fig 4.
Molecular docking results to Oye1 and NemA for trans-2-hexenal (T2H), trans-2-hexenoic acid (T2HA), 6-amino-trans-2-hexenoic acid (6H2A), and trans-2-hexenedioic acid (H2EA).
The distances between the α-carbon and hydroxyl hydrogen are indicated. Distances between the β-carbon and the N5 hydrogen of FMN are also shown. The angle is the N10-N5- β carbon angle. Measurements of the distances and angles are described in more detail in S7 Fig.
Fig 5.
SDS–page separation of the purified enzymes NemA (43 kDa) and Oye1 (48 kDa).
Fig 6.
Specific activities of NemA and Oye1 on target substrates and controls.
Units (U) are defined as μmol NADPH oxidized per min. All results are given as mean ± standard deviation of three replicates.
Fig 7.
Proposed reaction mechanism of Oye1 and NemA, visualized for the reduction trans-2-hexenal.
(Residues in brackets apply to NemA.) Upon hydrogen bonding of the aldehyde to the enzyme (hashed lines) electrons from the double bond are shifted towards the catalytic residues Asn194 (His182) and His191 (His185) (dotted lines), thereby creating a partial positive charge on the β-carbon (δ+) of the substrate, which activates the double bond and makes it prone to attack. When the double bond is activated the transfer of a hydride from the flavin N5 to the β-carbon of the substrate and protonation from Tyr196 (Tyr187) can occur, resulting in hexanal as the final product. The movement of electrons involved in the hydride attack and protonation are indicated by the curved arrows.
Fig 8.
Proposed reaction mechanism for Oye1 on α,β unsaturated carboxylic acid, here visualized with trans-2-hexenoic acid when the carboxylic acid is deprotonated (A) and when it is protonated (B). (A) The electrons of the additional negative charge are distributed between the two oxygens in a resonance structure (dotted lines). Upon hydrogen bonding with the catalytic residues of the enzyme, the electrons in the resonance structure will prevent activation of the unsaturated α,β bond and no reaction will occur. B) Upon hydrogen bonding to the enzyme (hashed lines) electrons from the double bond are shifted towards the catalytic residues Asn194 and His191 (dotted lines), thereby creating a partial positive charge (δ+) on the β-carbon of the substrate, which activates the double bond and makes it prone to attack. When the double bond is activated the transfer of a hydride from the flavin N5 to the β-carbon of the substrate and protonation from Tyr196 can occur, resulting in hexanoic acid as the final product. The movement of electrons involved in the hydride attack and protonation are indicated by the curved arrows.
Fig 9.
Activity of NemA and Oye1 in the presence of trans-2-hexenoic acid, 6-amino-trans-2-hexenoic acid, and trans-2-hexenedioic acid investigated for their inhibitory effect at concentrations up to 400 μM. Results are given as mean ± standard deviation of three replicates.
Fig 10.
Identification of ring closure product of 6-amino-trans-2-hexenoic acid.
NMR spectra of A) 6-amino-trans-2-hexenoic acid at 25°C before heating, B) 6-amino-trans-2-hexenoic acid after being heated at 90°C for 6 h, where additional peaks can be seen, indicated by asterisks. C) NMR spectrum of 2-pyrrolidineacetic acid. D) Proposed 2-pyrrolidineacetic acid formation mechanism by intra-molecular cyclization.
Fig 11.
Proposed mechanism for reduction of deprotonated carboxylic acid by Oye1.
Engineering of the enzyme by substitution of the native residues with putative X and Y residues could lead to the formation of hydrogen bonds between the enzyme binding pocket and both oxygens of the carboxylate group. Upon hydrogen bonding to the enzyme (hashed lines) electrons from the double bond are shifted towards the catalytic residues Asn194 and His191 for one of the oxygens, and to the residues X and Y for the other oxygen (dotted lines), thereby creating a partial positive charge on the β-carbon (δ+) of the substrate, which activates the double bond, making it prone to attack. When the double bond is activated the transfer of a hydride from the flavin N5 to the β-carbon of the substrate and protonation from Tyr196 can occur, resulting in hexanoic acid as the final product. The movement of electrons involved in the hydride attack and protonation are indicated by the curved arrows. For simplicity, only the mechanism for Oye1 is shown.