Role of a critical water in scytalone dehydratase-catalyzed reaction

Abstract

Scytalone dehydratase (EC 4.2.1.94) catalyzes the dehydration of two important intermediates in the biosynthesis of melanin, and it functions without metal ions or any cofactors. Using molecular orbital theory, we have examined the role of a critical water molecule in the mechanism of scytalone dehydratase. The water, together with an internal hydrogen bonding, contributes significantly to the stabilization of the transition state (or the enolate intermediate). The role of two active site tyrosines (Tyr-50 and Tyr-30) is (i) to hold the critical water in place so that it may stabilize the transition state without much structural rearrangement during the catalytic reaction, and (ii) to polarize the water, making it a better general acid. The stereochemistry of the scytalone dehydratase-catalyzed dehydration is also discussed.Rapid enzyme-catalyzed proton abstractions from carbon acids have attracted great interest. In view of the basicities of protein bases, these proton abstraction might be expected to be slow owning to low acidities of carbon acids. The mechanism by which enzymes accomplish such a difficult task has been a matter of continuing debate (1-4). One proposal invokes the formation of short strong hydrogen bonds during the catalytic process (2). This concept has been used to explain many enzymatic reactions. Our interest here is scytalone dehydratase.Scytalone dehydratase (EC 4.2.1.94) catalyzes the dehydration of two intermediates in the biosynthesis of melanin (Scheme 1) (5); it has been an important target for fungicides that control rice blast disease. Scytalone dehydratase is a trimeric enzyme consisting of three identical subunits, each unit having 129 amino acid residues. The x-ray crystal structure of scytalone dehydratase with a bound competitive inhibitor has been solved recently (6). Even at 2.9-Å resolution, two tightly bound water molecules were visible in the active site. On the basis of the crystallographic study, a catalytic mechanism was proposed as shown in Scheme 2 (6). According to this proposal, a water acts as a general acid to activate the carbonyl, making the ␣-C-H sufficiently acidic to be abstracted by a protein base, such as His-85, in the active site. As revealed by the x-ray crystallographic structure, the water is held in place by two tyrosine residues (Tyr-50 and Tyr-30). This dehydratase requires no metal ion or cofactors, so it is an especially interesting system.Recently, quantum mechanical methods have been used to examine enolization reactions pertaining to enzyme-catalyzed proton abstraction from weak carbon acids (7-9). Here we decided to employ quantum mechanical theory to investigate the role of the critical water in stabilizing the enolate intermediate and the role of these two active site tyrosine residues (Tyr-50 and Tyr-30) in scytalone dehydration catalyzed by scytalone dehydratase. THEORETICAL PROCEDUREAll calculations were carried out with the GAUSSIAN 94 program (10). For the ab initio molecular orbital calculations, geometry optimization was don...

High‐resolution structures of scytalone dehydratase‐inhibitor complexes crystallized at physiological pH

“…Tyr30 still forms a hydrogen bond with the active site water (the water implicated in catalysis). 25 The hydrogen bonding network involving the other active site water molecule (the putative product water molecule), 6 His85, His110, and the NH of 3 is very similar to that in the complexes with inhibitors 1 and 2. The polar methyl sulfoxide of 3 is located in a hydrophobic environment, flanked by Phe53.…”

“…Only small changes were found in the positions of the two active site water molecules, one of which (the one coordinated between His85 and His110) is believed to represent the product water molecule 6 and the other water molecule (the one coordinated between Tyr30 and Tyr50) is considered to have a catalytic role. 25 The finding that the four new structures have very similar positions for their active site hydrophilic residues and water molecules places constraints on our views of catalysis and inhibitor design. A recent review of ␤-elimination mechanisms suggests that SD catalyzes a syn elimination of water from scytalone.…”

Molecular and Biochemical Characterization of 2-Hydroxyisoflavanone Dehydratase. Involvement of Carboxylesterase-Like Proteins in Leguminous Isoflavone Biosynthesis

“…In fact, sitedirected mutagenesis of HIDH protein indicated that amino acid residues comprising the oxyanion hole and the catalytic triad are important for both HID and ester hydrolysis activities (Table III). Considering the established catalytic processes of carboxylesterase (Blow et al, 1969;Kraut, 1977) and microbial scytalone dehydratase, which is known to use a catalytic His residue for the dehydration of the substrate resembling 2-hydroxyisoflavanone (Lundqvist et al, 1994;Zheng and Bruice, 1998), a possible scheme for the HID reaction can be drawn, as in Figure 4. In this scheme, the nucleophilic oxygen of Thr in the catalytic triad acts as a base to abstract hydrogen from C-3 of the substrate, and the C-4 carbonyl is enolized to yield a negatively charged intermediate stabilized by the oxyanion hole.…”

Catalytic mechanism of scytalone dehydratase fromMagnaporthe grisea

“…The signiücance to catalysis of the crystallographic water ligand between Y30 and Y50 has been the subject of a rigorous analysis. 18 We have developed improved methods for obtaining X-ray quality crystals of SD complexed with a variety of inhibitors, yielding diþraction data sets as high as Several SD-inhibitor structures have 1.5 Ó. been solved with these methods, which provide crystals at a pH closer to physiological (pH 7.5 versus pH 5 in the original structure). Recently, Nakasako et al19 solved the X-ray structure of SD complexed with carpropamid by molecular replacement using the SD complex with the salicylamide inhibitor of Fig 2 as the basis.…”