File Name : figure s1.docx Caption : figure s1a. dcpip plate assay of cdh wt, w113a and y195f enzyme variants with cyclohexanone (1), 3-methylcyclohexanone (2), 4,4-dimethylcyclohexanone (3), δ-valerolactone (4), dihydrocoumarin (5), 3,3,5-trimethylcyclohexanone (6), 1-methyl-2-piperidone (7), δ-valerolactam (8), 3,4-dihydro-2(1h)-quinolinone (9) and no substrate (10). figure s1b. wt cdh assays. cyclohexanone (a1); 3-methylcyclohexanone (a2); 4,4-dimethylcyclohexanone (a3); δ-valerolactone (a4); dihydrocoumarin (a5); no substrate (a10). time h:mm:ss. figure s1c. w113a mutant assays. cyclohexanone (b1); 3-methylcyclohexanone (b2); 4,4-dimethylcyclohexanone (b3); δ-valerolactone (b4); dihydrocoumarin (b5); no substrate (b10). time h:mm:ss. figure s1d. y195f mutant assays. cyclohexanone (c1); 3-methylcyclohexanone (c2); 4,4-dimethylcyclohexanone (c3); δ-valerolactone (c4); dihydrocoumarin (c5); no substrate (c10). time h:mm:ss. File Name : figure s2.docx Caption : figure s2. hplc chromatograms from 5 mm standards of substrates (left) and corresponding products (right). chromatograms were recorded at 240 nm (top) and 275 nm (bottom) for each standard. chromatograms were acquired for 15-20 minutes. substrates generally absorbed weakly at 275 nm and products absorbed much more strongly at 240 nm by virtue of their double bonds. 3-methylcyclohexanone had a retention time of ~4.2 min and 3-methyl-2-cyclohexenone had a retention time of ~2.0 min (a). 4,4-dimethylcyclohexanone had a retention time of ~8.8 min and 4,4-dimethyl-2-cyclohexen-1-one had a retention time of ~5.6 min (b). File Name : figure s3.docx Caption : figure s3. hplc chromatograms showing incubation of cyclohexanone with dcpip and no enzyme (a), wt cdh enzyme (b) and the w113a variant (c). chromatograms were recorded at 240 nm and 275 nm. hplc analysis of cyclohexanone incubated with dcpip without enzyme revealed a peak for cyclohexanone at a retention time of 2 minutes and a peak for dcpip at ~30.1 min. when cyclohexanone was incubated with cdh wt or the w113a variant, the dcpip was reduced and therefore a change of retention time was observed from ~30.1 min to ~15.2 min and a peak for 2-cyclohexen-1-one was visible at ~1.4 min, confirming product formation. all retention times were compared with the retention times of the standards (figure s2). File Name : figure s4.docx Caption : figure s4. example hplc chromatograms showing products of 3-methylcyclohexanone (a), 4,4-dimethylcyclohexanone (b) with either wt cdh or the w113a variant. chromatograms were recorded at 240 nm and 275 nm. all retention times were compared with the retention times of the standards (figure s2). File Name : figure s5.docx Caption : figure s5. cdh catalyses the production of 5-methyl-2-cyclohexen-1-one. 1h nmr spectra of 26 nm cdh (red; top trace) and 26 µm cold-denatured cdh (blue; bottom trace) incubated with 1 mm 3-methylcyclohexanone for 1 hour at 25°c. inset shows detail of feature at c. 7.15 ppm, which corresponds to the proton highlighted in the structure of 5-methyl-2-cyclohexen-1-one. this feature is entirely absent in the cold-denatured spectrum. it has the characteristic ddd (j (hz) = 9.93, 5.83, 2.96) shape expected for the 5-methyl isomer and would be absent in the case that cdh catalysed the production of the 3-methyl isomer. File Name : figure s6.docx Caption : figure s6. superimposition of 3-ketosteroid δ1-dehydrogenase from rhodococcus erythropolis sq1, pdb 4c3x(1) with wildtype cdh using coot(2). rmsd achieved: 1.8823ᾰ. the overlaid structures were drawn using pymol. 1. a. rohman, n. van oosterwijk, a.-m. w. h. thunnissen, b. w. dijkstra, crystal structure and site-directed mutagenesis of 3-ketosteroid δ1-dehydrogenase from rhodococcus erythropolis sq1 explain its catalytic mechanism*. journal of biological chemistry 288, 35559-35568 (2013). 2. p. emsley, k. cowtan, coot: model-building tools for molecular graphics. acta crystallogr d biol crystallogr 60, 2126-2132 (2004). File Name : figure s7.docx Caption : figure s7. fad (purple sticks) and cdh (gold cartoon) form an unusually short intermolecular contact. the sγ atom of cys305 (gold sticks) is situated only 1.84 å from the c8m atom of fad. electron density at 2.0 σ (black mesh) bridges this gap, indicating a degree of shared electrons. the complexed form is shown; the intermolecular distance was identical in the non-complexed form. File Name : figure s8.docx Caption : figure s8. dimer orientation in cdh. panel a. dimer orientation in wt apo enzyme. chain a is shown as teal cartoon in standard orientation; chain b is shown as green surface with intermolecular interface shown in red. monomers orient in head-to-head conformation. approximate position of dimer active sites shown as asterisks. panel b. cdh y195f mutant in complex with cyclohexanone, surface representation as in a. chain a shown in gold; chain b shown in yellow with interface shown in red panel c. the α10 loop res (375-383) showing numerous intermolecular polar contacts. residues forming hydrogen bonds shown as sticks; hydrogen bonds shown as yellow dashed lines. File Name : figure s9.docx Caption : figure s9. cdh and cdh-y195f·a2q have been solved to atomic resolution a. the apo form of cdh (teal cartoon) has a solvent-filled channel (the entrance) leading into the active site, which is formed as a cleft between the two domains. the inlet of the channel (black dashed circle) is wide enough to accommodate the passage of one molecule. the outlet of the channel (red dashed circle; the exit) is not wide enough to pass the substrate but is instead filled with well-structured waters, which possibly act as a reservoir of protons and electrons to reoxidise fadh (purple sticks) after catalysis. b. upon binding three molecules (mauve sticks; labelled a2q1-3) the holo form of cdh y195f (gold cartoon) undergoes subtle interdomain movement (indicated by black arrows). the binding of a2q1 into the active site triggers the closure of the inlet (figure 2). a2q2 binds in the shallow pocket left after the closure of the inlet, but the occupancy is poor, and this binding event is unlikely to be functional. a2q3 binds in a shallow pocket distal from the active site, and is probably a crystallographic artefact. c. the active site of cdh y195f with 2-cyclohexen-1-one bound. a2q1 stacks against the isoalloxazine ring of fad, oriented in the correct position for dehydrogenation by the four catalytic residues (gold sticks) in concert with fad. hydrogen bonds are shown as yellow dashed lines; redox transfers are shown as green dashed lines. structures were drawn with pymol (delano scientific www.pymol.org). File Name : figure s10.docx Caption : figure s10. the rmsd obtained from wild type cdh and w113a in complex with cyclohexanone ligand for 500 ns trajectory. the rmsd values of cα atoms of the protein and the heavy atoms of cyclohexanone ligand are shown in blue and orange colours respectively. File Name : figure s11.docx Caption : figure s11. the hydrogen bonding in the wild type cdh and w113a in complex with cyclohexanone. the interatomic distance between the carbonyl oxygen of the cyclohexanone and the side chain of y570 and the backbone of g574 residue for the 500 ns trajectory. File Name : figure s12.docx Caption : figure s12. the interatomic distance of the axial proton located at the cα and cβ position of cyclohexanone in wt and w113a for the 500 ns trajectory.