Crystal Structures of Two Aromatic Hydroxylases Involved in the Early Tailoring Steps of Angucycline Biosynthesis

Koskiniemi, H.; Metsä‐Ketelä, Mikko; Dobritzsch, Doreen; Kallio, Pauli T.; Korhonen, Hanna; Mäntsälä, Pekka; Schneider, Günter; Niemi, Jarmo

doi:10.1016/j.jmb.2007.06.087

Cited by 61 publications

(100 citation statements)

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“…Reactions catalyzed by two-component monooxygenases include oxygenation and halogenation of organic compounds such as p-hydroxyphenylacetate (9), phenol (10), trichorophenol (11,12), p-nitrophenol (13), styrene (14 -16), alkane sulfonate (17,18), and EDTA (19). Two-component monooxygenases are also involved in oxygenation and halogenation reactions in the biosynthetic pathways of actinorhodin (ActVA) (20), angucyclin (21), enediyne (SgcC) (22), rebeccamycin (RebH) (23), pyrrolnitrin (PrnA) (24), violacein (25), kutzneride (26), and differentiation-inducing factor-1 (27).…”

Section: ؊1 Smentioning

confidence: 99%

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Ruangchan

Tongsook

Sucharitakul

et al. 2011

Journal of Biological Chemistry

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Flavin-dependent monooxygenases catalyze the incorporation of a single atom of molecular oxygen into organic substrates (1, 2). The enzymes have been classified into six classes according to their catalytic and structural properties. They have also been categorized into two major types according to their protein components: a single-component type, in which reduction of a flavin cofactor and oxygenation of an organic substrate occurs within the same single polypeptide chain; and a two-component type, in which each reaction occurs on separate proteins (1, 2). Single-component monooxygenases have been identified since the 1960s and have been found to be involved in the aerobic metabolism of aromatic and aliphatic compounds in various organisms (2-4). The well known prototype for single-component monooxygenases is p-hydroxybenzoate hydroxylase from Pseudomonas fluorescens (5, 6). The first enzyme identified as a two-component monooxygenase was bacterial luciferase (7). Nevertheless, most of the two-component monooxygenases have been identified only during the past decade and have increasingly emerged as common enzymes in nature that are involved in many important reactions in various microorganisms (1,8). Reactions catalyzed by two-component monooxygenases include oxygenation and halogenation of organic compounds such as p-hydroxyphenylacetate (9), phenol (10), trichorophenol (11, 12), p-nitrophenol (13), styrene (14 -16), alkane sulfonate (17, 18), and EDTA (19). Two-component monooxygenases are also involved in oxygenation and halogenation reactions in the biosynthetic pathways of actinorhodin (ActVA) (20), angucyclin (21), enediyne (SgcC) (22), rebeccamycin (RebH) (23), pyrrolnitrin (PrnA) (24), violacein (25), kutzneride (26), and differentiation-inducing factor-1 (27).All flavin-dependent monooxygenases perform oxygenation through the participation of a reactive intermediate, C4a-hydroperoxy-flavin, which has been well documented and detected by transient kinetics for the reactions of singlecomponent monooxygenases. These monooxygenases include p-hydroxybenzoate hydroxylase (PHBH) 3 (5, 6), phenol hydroxylase (28), melilotate hydroxylase (29), antranilate hydroxylase (30), Baeyer-Villiger monooxygenases (31, 32), 2-methyl-3-hydroxypyridine-5-carboxylic oxygenase * This work was supported by Grants BRG5180002 (to P. C.) and MRG5380240 (to J. S.) from the Thailand Research Fund and a grant from the Faculty of Science, Mahidol University (to P. C.

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Section: ؊1 Smentioning

confidence: 99%

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Ruangchan

Tongsook

Sucharitakul

et al. 2011

Journal of Biological Chemistry

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“…Structural and rapid kinetics studies on wild-type and engineered enzyme variants elucidated many details of the catalytic cycle of PHBH. Moreover, these investigations revealed that the isoalloxazine ring of the FAD cofactor is mobile and can swing in and out of [19,20] A 1PN0; 1FOH phenol 2-monooxygenase (PHHY) [22,23] A 2DKH; 2DKI 3-hydroxybenzoate 4-hydroxylase (MHBH) [24] A 2 A1; 2 A2 UW16 12-hydroxylase (PgaE/CabE) [25] A 2R0C; 2R0G; 2R0P 7-carboxy-K252c hydroxylase (RebC) [26] A 2VOU 2,6-dihydroxypyridine 3-hydroxylase (DHP) [27] A 2RGJ phenazine-1-carboxylate hydroxylase (PhzS) [28] A 3IHG aklavinone 11-hydroxylase (RdmE) [29] A 3GMB; 3GMC 2-methyl-3-hydroxypyridine-5-carboxylic acid oxygenase (MHPCO) [30] E 3IHM styrene monooxygenase (StyA) [9] Scheme 1. Reactions catalyzed by (A) PHBH, (B) PHHY, (C) MHBH, (D) DHP hydroxylase.…”

Section: Introductionmentioning

confidence: 99%

Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases

et al. 2011

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Monooxygenases perform chemo-, regioand/or enantioselective oxygenations of organic substrates under mild reaction conditions. These properties and the increasing number of representatives along with effective preparation methods place monooxygenases in the focus of industrial biocatalysis. Mechanistic and structural insights reveal reaction sequences and allow turning them into efficient tools for the production of valuable products. Herein we describe two biocatalytically relevant subclasses of flavoprotein monooxygenases with a close evolutionary relation: subclass A represented by p-hydroxybenzoate hydroxylase (PHBH) and subclass E formed by styrene monooxygenases (SMOs). PHBH family members perform highly regioselective hydroxylations on a wide variety of aromatic compounds. The more recently discovered SMOs catalyze a number of stereoselective epoxidation and sulfoxidation reactions. Mechanistic and structural studies expose distinct characteristics, which provide a promising source for future biocatalyst development.

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“…Only in the past decade have two-component monooxygenases received more attention (1)(2)(3)(4). They have been found to catalyze flavin reduction and substrate oxidation using separate polypeptides and are important for the metabolism of aromatic and aliphatic compounds in bacteria (4 -7); biosynthetic pathways of antibiotics and cancer drugs, such as actinorhodin (8), rebeccamycin (9), violacein (10), enediyne (11), angucycline (12), kijanimicin (13), and kutzneride (14); and bacterial pathogenesis (15). During the past few years, several crystal structures of the oxygenase components of these enzymes have been reported, including HpaB from Thermus thermophilus HB8 (16), LadA from Geobacillus thermodenitrificans NG80 -2 (17), RebH from Lechevalieria aerocolonigenes (18,19), TftD from Burkholderia cepacia AC1100 (20), SMOA from Pseudomonas putida S12 (21), HsaA from Mycobacterium tuberculosis (15), KijD3 from Actinomadura kijaniata (22), and ORF36 from Micromonospora carbonacea var.…”

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confidence: 99%

Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

Thotsaporn

Chenprakhon

Sucharitakul

et al. 2011

Journal of Biological Chemistry

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p-Hydroxyphenylacetate (HPA) 3-hydroxylase is a two-component flavin-dependent monooxygenase. Based on the crystal structure of the oxygenase component (C 2 ), His-396 is 4.5 Å from the flavin C4a locus, whereas Ser-171 is 2.9 Å from the flavin N5 locus. We investigated the roles of these two residues in the stability of the C4a-hydroperoxy-FMN intermediate. The results indicated that the rate constant for C4a-hydroperoxy-FMN formation decreased ϳ30-fold in H396N, 100-fold in H396A, and 300-fold in the H396V mutant, compared with the wild-type enzyme. Lesser effects of the mutations were found for the subsequent step of H 2 O 2 elimination. Studies on pH dependence showed that the rate constant of H 2 O 2 elimination in H396N and H396V increased when pH increased with pK a >9.6 and >9.7, respectively, similar to the wild-type enzyme (pK a >9.4). These data indicated that His-396 is important for the formation of the C4a-hydroperoxy-FMN intermediate but is not involved in H 2 O 2 elimination. Transient kinetics of the Ser-171 mutants with oxygen showed that the rate constants for the H 2 O 2 elimination in S171A and S171T were ϳ1400-fold and 8-fold greater than the wild type, respectively. Studies on the pH dependence of S171A with oxygen showed that the rate constant of H 2 O 2 elimination increased with pH rise and exhibited an approximate pK a of 8.0. These results indicated that the interaction of the hydroxyl group side chain of Ser-171 and flavin N5 is required for the stabilization of C4a-hydroperoxy-FMN. The double mutant S171A/H396V reacted with oxygen to directly form the oxidized flavin without stabilizing the C4a-hydroperoxy-FMN intermediate, which confirmed the findings based on the single mutation that His-396 was important for formation and Ser-171 for stabilization of the C4a-hydroperoxy-FMN intermediate in C 2 .

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Crystal Structures of Two Aromatic Hydroxylases Involved in the Early Tailoring Steps of Angucycline Biosynthesis

Cited by 61 publications

References 57 publications

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

pH-dependent Studies Reveal an Efficient Hydroxylation Mechanism of the Oxygenase Component of p-Hydroxyphenylacetate 3-Hydroxylase

Catalytic and Structural Features of Flavoprotein Hydroxylases and Epoxidases

Stabilization of C4a-Hydroperoxyflavin in a Two-component Flavin-dependent Monooxygenase Is Achieved through Interactions at Flavin N5 and C4a Atoms

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