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.

plex, which in turn promotes oxygen atom transfer via an electrophilic aromatic substitution mechanism. Analysis of Ser-146 variants revealed that this residue is necessary for but not directly engaged in hydroxylation. Product formation in S146A is pH-independent and constant at ϳ70% over a pH range of 6 -10, whereas product formation for S146C decreased from ϳ65% at pH 6.0 to 27% at pH 10.0. These data indicate that the ionization of Cys-146 in the S146C variant has an adverse effect on hydroxylation, possibly by perturbing formation of the His ␦؉ ⅐HPA ␦؊ complex needed for hydroxylation.Incorporation of single oxygen atoms into organic compounds (monooxygenation) by hydroxylation or epoxidation is an important biological process for aerobic organisms. The monooxygenation reaction is generally catalyzed by cytochrome P450, metalloenzymes, pterin-dependent and flavindependent monooxygenases (1). Flavin-dependent monooxygenases are involved in a wide variety of biological processes (2, 3). These enzymes catalyze the monooxygenation of many aromatic and aliphatic compounds (3).Based on the number of proteins required for catalysis, flavin-dependent monooxygenases can be classified into singleprotein component and two-protein component types (2-5). Besides an organic substrate, both types of enzymes require NAD(P)H and oxygen as co-substrates. The initial part of the reaction is the reduction of an enzyme-bound flavin by NAD(P)H to generate reduced flavin followed by the reaction of reduced flavin with oxygen to form the C4a-(hydro)peroxy flavin, a key intermediate that is required for oxygenation of an organic substrate. Oxygenation occurs via an oxygen atom transfer from C4a-hydroperoxy flavin to an organic substrate, resulting in the formation of a C4a-hydroxy flavin intermediate and an oxygenated product. The C4a-hydroxy flavin dehydrates at the last step of the reaction to form the final species, oxidized flavin (2-4, 6). All steps for the reactions of singleprotein component flavin-dependent monooxygenases occur within the same polypeptide, whereas for the two-protein component type the flavin reduction occurs on a reductase component and the oxygenation occurs on an oxygenase component (4 -8). The mechanism by which the reduced flavin is transferred involves simple diffusion or protein-protein contacts (5,8). Although single-component flavin-dependent monooxygenases have been studied for more than 40 years, two-component flavin-dependent monooxygenases have only received significant attention during the past decade after recent discoveries of their involvement in a wide variety of reactions (2, 5).The mechanism by which the oxygen atom transfer occurs in flavin-dependent monooxygenases is well understood for only a few enzymatic systems. The best understood oxygenation reaction is the hydroxylation of aromatic compounds catalyzed *

Background: 3-Hydroxybenzoate 6-hydroxylase (3HB6H) is a flavoprotein hydroxylase catalyzing the para-hydroxylation of 3-hydroxybenzoate. Results: The oxidative half-reaction was studied using transient kinetics. The enzyme reaction mechanism was elucidated. Conclusion:The product release and the hydroxylation limit the turnovers and the enzyme shows characteristics different from the ortho-hydroxylation enzymes. Significance: This is the first report on the oxygenation mechanism of a para-hydroxylating flavoenzyme.

We describe the characterization of a gene for mild nonsyndromic autosomal recessive intellectual disability (ID) in two unrelated families, one from Austria, the other from Pakistan. Genome-wide single nucleotide polymorphism microarray analysis enabled us to define a region of homozygosity by descent on chromosome 17q25. Whole-exome sequencing and analysis of this region in an affected individual from the Austrian family identified a 5 bp frameshifting deletion in the METTL23 gene. By means of Sanger sequencing of METTL23, a nonsense mutation was detected in a consanguineous ID family from Pakistan for which homozygosity-by-descent mapping had identified a region on 17q25. Both changes lead to truncation of the putative METTL23 protein, which disrupts the predicted catalytic domain and alters the cellular localization. 3D-modelling of the protein indicates that METTL23 is strongly predicted to function as an S-adenosyl-methionine (SAM)-dependent methyltransferase. Expression analysis of METTL23 indicated a strong association with heat shock proteins, which suggests that these may act as a putative substrate for methylation by METTL23. A number of methyltransferases have been described recently in association with ID. Disruption of METTL23 presented here supports the importance of methylation processes for intact neuronal function and brain development.

The oxygenase component (C) of p-hydroxyphenylacetate (4-HPA) 3-hydroxylase (HPAH) from Acinetobacter baumannii catalyzes the hydroxylation of various phenolic acids. In this report, we found that substitution of a residue close to the phenolic group binding site to yield the S146A variant resulted in an enzyme that is more effective than the wild-type in catalyzing the hydroxylation of 4-aminophenylacetate (4-APA). Product yields for both wild-type and S146A enzymes are better at lower pH values. Multiple turnover reactions of the wild-type and S146A enzymes indicate that both enzymes first hydroxylate 3-APA to give 3-hydroxy-4-aminophenylacetate (3-OH-4-APA), which is further hydroxylated to give 3,5-dihydroxy-4-aminophenylacetate, similar to the reaction of C with 4-HPA. Stopped-flow experiments showed that 4-APA can only bind to the wild-type enzyme at pH 6.0 and not at pH 9.0, while it can bind to S146A under both pH conditions. Rapid-quench flow results indicate that the wild-type enzyme has low reactivity toward 4-APA hydroxylation, with a hydroxylation rate constant (k) for 4-APA of 0.028 s compared to 17 s for 4-HPA, the native substrate. In contrast, for S146A, the hydroxylation rate constants for both substrates are very similar (2.6 s for 4-HPA versus 2.5 s for 4-APA). These data indicate that Ser146 is a key catalytic residue involved in optimizing C reactivity toward a phenolic compound. Removing this hydroxyl group expands C activity toward a non-natural aniline substrate. This understanding should be helpful for future rational engineering of other two-component flavin-dependent monooxygenases that have this conserved Ser residue.