Catalytic Mechanism of Scytalone Dehydratase:  Site-Directed Mutagenisis, Kinetic Isotope Effects, and Alternate Substrates

Abstract

On the basis of the X-ray crystal structure of scytalone dehydratase complexed with an active center inhibitor [Lundqvist, T., Rice, J., Hodge, C. N., Basarab, G. S., Pierce, J. and Lindqvist, Y. (1994) Structure (London) 2, 937-944], eight active-site residues were mutated to examine their roles in the catalytic mechanism. All but one residue (Lys73, a potential base in an anti elimination mechanism) were found to be important to catalysis or substrate binding. Steady-state kinetic parameters for the mutants support the native roles for the residues (Asn131, Asp31, His85, His110, Ser129, Tyr30, and Tyr50) within a syn elimination mechanism. Relative substrate specificities for the two physiological substrates, scytalone and veremelone, versus a Ser129 mutant help assign the orientation of the substrates within the active site. His85Asn was the most damaging mutation to catalysis consistent with its native roles as a general base and a general acid in a syn elimination. The additive effect of Tyr30Phe and Tyr50Phe mutations in the double mutant is consistent with their roles in protonating the substrate's carbonyl through a water molecule. Studies on a synthetic substrate, which has an anomeric carbon atom which can better stabilize a carbocation than the physiological substrate (vermelone), suggest that His110Asn prefers this substrate over vermelone in order to balance the mutation-imposed weakness in promoting the elimination of hydroxide from substrates. All mutant enzymes bound a potent active-site inhibitor in near 1:1 stoichiometry, thereby supporting their active-site integrity. An X-ray crystal structure of the Tyr50Phe mutant indicated that both active-site waters were retained, likely accounting for its residual catalytic activity. Steady-state kinetic parameters with deuterated scytalone gave kinetic isotope effects of 2.7 on kcat and 4.2 on kcat/Km, suggesting that steps after dehydration partially limit kcat. Pre-steady-state measurements of a single-enzyme turnover with scytalone gave a rate that was 6-fold larger than kcat. kcat/Km with scytalone has a pKa of 7.9 similar to the pKa value for the ionization of the substrate's C6 phenolic hydroxyl, whereas kcat was unaffected by pH, indicating that the anionic form of scytalone does not bind well to enzyme. With an alternate substrate having a pKa above 11, kcat/Km had a pKa of 9.3 likely due to the ionization of Tyr50. The non-enzyme-catalyzed rate of dehydration of scytalone was nearly a billion-fold slower than the enzyme-catalyzed rate at pH 7.0 and 25 degrees C. The non-enzyme-catalyzed rate of dehydration of scytalone had a deuterium kinetic isotope effect of 1.2 at pH 7.0 and 25 degrees C, and scytalone incorporated deuterium from D2O in the C2 position about 70-fold more rapidly than the dehydration rate. Thus, scytalone dehydrates through an E1cb mechanism off the enzyme.

Crystal structure and functional analysis of tetracenomycin ARO/CYC: Implications for cyclization specificity of aromatic polyketides

“…This is similar to the Ser and Asn proposed to bind the C6-and C8-OH groups of scytalone (20, Fig. S4) in scytalone dehydratase studies (12). This conformation also docks C1-C8 and C15-C20 of the polyketide chain in the pocket and positions the C1 thiol at the pocket entrance, where ACP would be covalently linked to C1 via a thioester bond.…”

Hydroxyl regioisomerization of anthracycline catalyzed by a four-enzyme cascade

“…Further analysis indicates that KstA10 is homologous to NAD + -dependent nucleotide diphosphate sugar epimerases (23), which act on totally different substrates from that in KST biosynthesis. In addition, scytalone dehydratase in the fungal melanin biosynthesis is cofactor independent (18,19). However, evidence obtained from our biochemical assays suggested that the dehydration catalyzed by KstA10 is dependent on NAD(P)H (SI Appendix, Fig.…”

“…Our in vitro results unequivocally demonstrated that KstA15/A16 synergistically catalyze hydroxylation of 3 to produce 5, which may serve as an intermediate during the hydroxyl regioisomerization process; thus, a dehydroxylation must be involved in the transformation from 5 to 4. To the best of our knowledge, two enzymatic dehydroxylation pathways of aromatic ring had been reported: Birch-like one electron reduction mechanism, as exemplified by 4-hydroxybenzoyl-CoA reductase, which is a Mo-flavo-Fe/S-dependent protein (16,17), and reduction-dehydration process-mediated dehydroxylation in the fungal melanin biosynthetic pathway, operated by 1,3,6,8-tetradroxynaphthalene reductase, 1,3,8-tridroxynaphthalene reductase, both belonging to the short-chain dehydrogenase/reductase (SDR) superfamily, and scytalone dehydratase (18)(19)(20). However, bioinformatic analysis revealed that neither Mo-flavo-Fe/S protein nor scytalone dehydratase homolog has been found in the KST pathway.…”

Two Novel Classes of Enzymes Are Required for the Biosynthesis of Aurofusarin in Fusarium graminearum

“…The ligand-binding pocket of the enzyme is a cone-shaped ␣ϩ␤ barrel, consisting of eight ␤ strands and four ␣ helixes sandwiched together. The enzyme acts independently of cofactors, prosthetic groups, or metal ions (37). In addition to its natural substrates, scytalone and vermelone, SD is also able to act on the pyrone 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO), which resembles the structure of YWA1 (38).…”

“…The MIPS annotation of AurS includes the prediction of a transmembrane domain located from positions 7 to 29, and an analysis using SignalP suggests that this N-terminal sequence contains a signal peptide, which is cleaved between Ser 36 and Gln 37 . This combined with a TargetP prediction suggests that the signal peptide induces secretion of the protein to the extracellular surface of the cell (score: 0.899).…”