Roles of Substrate Distortion and Intramolecular Hydrogen Bonding in Enzymatic Catalysis by Scytalone Dehydratase

Abstract

Alternative substrates and site-directed mutations of active-site residues are used to probe factors controlling the catalytic efficacy of scytalone dehydratase. In the E1cb-like, syn-elimination reactions catalyzed, efficient catalysis requires distortion of the substrate ring system to facilitate proton abstraction from its C2 methylene and elimination of its C3 hydroxyl group. Theoretical calculations indicate that such distortions are more readily achieved in the substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one (DDBO) than in the physiological substrates vermelone and scytalone by approximately 2 kcal/mol. A survey of 12 active-site amino acid residues reveals 4 site-directed mutants (H110N, N131A, F53A, and F53L) have higher relative values of k(cat) and k(cat)/K(m) for DDBO over scytalone and for DDBO over vermelone than the wild-type enzyme, thus suggesting substrate-distortion roles for the native residues in catalysis. A structural link for this function is in the modeled enzyme-substrate complex where F53 and H110 are positioned above and below the substrate's C3 hydroxyl group, respectively, for pushing and pulling the leaving group into the axial orientation of a pseudo-boat conformation; N131 hydrogen-bonds to the C8 hydroxyl group at the opposite end of the substrate, serving as a pivot for the actions of F53 and H110. Deshydroxyvermelone lacks the phenolic hydroxyl group and the intramolecular hydrogen bond of vermelone. The relative values of k(cat) (95) and k(cat)/K(m) (1800) for vermelone over deshydroxyvermelone for the wild-type enzyme indicate the importance of the hydroxyl group for substrate recognition and catalysis. Off the enzyme, the much slower rates for the solvolytic dehydration of deshydroxyvermelone and vermelone are similar, thus specifying the importance of the hydroxyl group of vermelone for enzyme catalysis.

Energy of Intramolecular Hydrogen Bonding in ortho-Hydroxybenzaldehydes, Phenones and Quinones. Transfer of Aromaticity from ipso-Benzene Ring to the Enol System(s)

“…Therefore, it is important to recognize the role played by the HB in stabilizing radicals and the anionic species in phenols, for example, in acylphloroglucinols [ 32 , 33 ]. Usinic acid [ 34 ] produced by lichens acts as an effective antibiotic and scytalone dehydratase is crucial for the fungal melanin biosynthetic pathway [ 35 ]. Some quinones (lapachol, caryopteron) [ 36 ] exhibit important cytotoxic activity against cancer cells, naphthazarin [ 37 ] acts as an antibacterial agent, apoptosis inducer, antineoplastic agent, and the most common application of anthraquinones remains the dyeing of both natural and synthetic fibres [ 38 ].…”

β-d-Xylosidase from Selenomonas ruminantium: Role of Glutamate 186 in Catalysis Revealed by Site-Directed Mutagenesis, Alternate Substrates, and Active-Site Inhibitor

“…In combination with site-directed mutagenesis, substrates with different physical properties, relevant to the reaction coordinate, are generally useful in pinpointing roles of native amino acid residues in stabilizing different aspects of the reaction's transition state. This strategy was first applied to identify active-site residues involved in distorting substrate ring conformations to their transition-state geometries mandated by stereoelectronic principles in the case of scytalone dehydratase acting on scytalone and the more easily deformed alternate substrate 2,3-dihydro-2,5-dihydroxy-4H-benzopyran-4-one [21]. The strategy was first applied to glycoside hydrolases in the case of SXA acting on 4NPX and 4NPA [11].…”

Enhancing the Stress Tolerance and Virulence of an Entomopathogen by Metabolic Engineering of Dihydroxynaphthalene Melanin Biosynthesis Genes

“…Moreover, our results indicated that only incorporation of PKS, SCD, and THR cloned from A. alternata into the genome of M. anisopliae allowed proper function by promoting the synthesis of the desired DHN melanins. This makes sense based on the recent enzymatic kinetic and protein crystallographic studies on two naphthol reductases (4HNR and 3HNR) and SCD (32,56,64). The two enzymes 4HNR and 3HNR have been shown to catalyze the reactions of two physiological substrates, 1,3,6,8-THN and 1,38-THN, but the substrate preferences and K cat /K m ratios vary (32,56).…”

“…The two enzymes 4HNR and 3HNR have been shown to catalyze the reactions of two physiological substrates, 1,3,6,8-THN and 1,38-THN, but the substrate preferences and K cat /K m ratios vary (32,56). Likewise, the capability of SCD to catalyze the reaction of the two physiological substrates, scytalone and vermelone, has been unequivocally proven (64).…”

“…Finally, the intermediate 1,8-dihydroxynaphthalene (1, is polymerized into melanin by the catalysis of phenol oxidase, peroxidase, laccase, or catalase ( Fig. 1A) (3,8,32,56,64).…”