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Histone deacetylase (HDAC) inhibitors are proven anticancer therapeutics and have potential in the treatment of many other diseases including HIV infection, Alzheimer's disease, and Friedreich's ataxia. A problem with the currently available HDAC inhibitors is that they have limited specificity and target multiple deacetylases. Designing isoform-selective inhibitors has proven challenging due to similarities in the structure and chemistry of HDAC active sites. However, the fact that HDACs 1, 2, and 3 are recruited to several large multi-subunit complexes, each with particular biological functions, raises the possibility of specifically inhibiting individual complexes. This may be assisted by recent structural and functional information about the assembly of these complexes. Here, we review the available structural information and discuss potential targeting strategies., (Crown Copyright © 2016. Published by Elsevier Ltd. All rights reserved.)

Context: Isolated congenital central hypothyroidism (CeH) can result from mutations in TRHR, TSHB, and IGSF1, but its etiology often remains unexplained. We identified a missense mutation in the transducin β-like protein 1, X-linked (TBL1X) gene in three relatives diagnosed with isolated CeH. TBL1X is part of the thyroid hormone receptor-corepressor complex., Objective: The objectives of the study were the identification of TBL1X mutations in patients with unexplained isolated CeH, Sanger sequencing of relatives of affected individuals, and clinical and biochemical characterization; in vitro investigation of functional consequences of mutations; and mRNA expression in, and immunostaining of, human hypothalami and pituitary glands., Design: This was an observational study., Setting: The study was conducted at university medical centers., Patients: Nineteen individuals with and seven without a mutation participated in the study., Main Outcome Measures: Outcome measures included sequencing results, clinical and biochemical characteristics of mutation carriers, and results of in vitro functional and expression studies., Results: Sanger sequencing yielded five additional mutations. All patients (n = 8; six males) were previously diagnosed with CeH (free T 4 [FT4] concentration below the reference interval, normal thyrotropin). Eleven relatives (two males) also carried mutations. One female had CeH, whereas 10 others had low-normal FT4 concentrations. As a group, adult mutation carriers had 20%-25% lower FT4 concentrations than controls. Twelve of 19 evaluated carriers had hearing loss. Mutations are located in the highly conserved WD40-repeat domain of the protein, influencing its expression and thermal stability. TBL1X mRNA and protein are expressed in the human hypothalamus and pituitary., Conclusions: TBL1X mutations are associated with CeH and hearing loss. FT4 concentrations in mutation carriers vary from low-normal to values compatible with CeH.

Drugs that recapitulate aspects of the exercise adaptive response have the potential to provide better treatment for diseases associated with physical inactivity. We previously observed reduced skeletal muscle class IIa HDAC (histone deacetylase) transcriptional repressive activity during exercise. Here, we find that exercise-like adaptations are induced by skeletal muscle expression of class IIa HDAC mutants that cannot form a corepressor complex. Adaptations include increased metabolic gene expression, mitochondrial capacity, and lipid oxidation. An existing HDAC inhibitor, Scriptaid, had similar phenotypic effects through disruption of the class IIa HDAC corepressor complex. Acute Scriptaid administration to mice increased the expression of metabolic genes, which required an intact class IIa HDAC corepressor complex. Chronic Scriptaid administration increased exercise capacity, whole-body energy expenditure and lipid oxidation, and reduced fasting blood lipids and glucose. Therefore, compounds that disrupt class IIa HDAC function could be used to enhance metabolic health in chronic diseases driven by physical inactivity., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Metallo-β-lactamases catalyze the hydrolysis of most β-lactam antibiotics and hence represent a major clinical concern. The development of inhibitors for these enzymes is complicated by the diversity and flexibility of their substrate-binding sites, motivating research into their structure and function. In this study, we examined the conformational properties of the Bacillus cereus β-lactamase II in the presence of chemical denaturants using a variety of biochemical and biophysical techniques. The apoenzyme was found to unfold cooperatively, with a Gibbs free energy of stabilization (ΔG(0)) of 32 ± 2 kJ·mol(-1) For holoBcII, a first non-cooperative transition leads to multiple interconverting native-like states, in which both zinc atoms remain bound in an apparently unaltered active site, and the protein displays a well organized compact hydrophobic core with structural changes confined to the enzyme surface, but with no catalytic activity. Two-dimensional NMR data revealed that the loss of activity occurs concomitantly with perturbations in two loops that border the enzyme active site. A second cooperative transition, corresponding to global unfolding, is observed at higher denaturant concentrations, with ΔG(0) value of 65 ± 1.4 kJ·mol(-1) These combined data highlight the importance of the two zinc ions in maintaining structure as well as a relatively well defined conformation for both active site loops to maintain enzymatic activity., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: The combination of developmental delay, facial characteristics, hearing loss and abnormal fat distribution in the distal limbs is known as Pierpont syndrome. The aim of the present study was to detect and study the cause of Pierpont syndrome., Methods: We used whole-exome sequencing to analyse four unrelated individuals with Pierpont syndrome, and Sanger sequencing in two other unrelated affected individuals. Expression of mRNA of the wild-type candidate gene was analysed in human postmortem brain specimens, adipose tissue, muscle and liver. Expression of RNA in lymphocytes in patients and controls was additionally analysed. The variant protein was expressed in, and purified from, HEK293 cells to assess its effect on protein folding and function., Results: We identified a single heterozygous missense variant, c.1337A>G (p.Tyr446Cys), in transducin β-like 1 X-linked receptor 1 (TBL1XR1) as disease-causing in all patients. TBL1XR1 mRNA expression was demonstrated in pituitary, hypothalamus, white and brown adipose tissue, muscle and liver. mRNA expression is lower in lymphocytes of two patients compared with the four controls. The mutant TBL1XR1 protein assembled correctly into the nuclear receptor corepressor (NCoR)/ silencing mediator for retinoid and thyroid receptors (SMRT) complex, suggesting a dominant-negative mechanism. This contrasts with loss-of-function germline TBL1XR1 deletions and other TBL1XR1 mutations that have been implicated in autism. However, autism is not present in individuals with Pierpont syndrome., Conclusions: This study identifies a specific TBL1XR1 mutation as the cause of Pierpont syndrome. Deletions and other mutations in TBL1XR1 can cause autism. The marked differences between Pierpont patients with the p.Tyr446Cys mutation and individuals with other mutations and whole gene deletions indicate a specific, but as yet unknown, disease mechanism of the TBL1XR1 p.Tyr446Cys mutation., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Histone deacetylases (HDACs) 1, 2 and 3 form the catalytic subunit of several large transcriptional repression complexes. Unexpectedly, the enzymatic activity of HDACs in these complexes has been shown to be regulated by inositol phosphates, which bind in a pocket sandwiched between the HDAC and co-repressor proteins. However, the actual mechanism of activation remains poorly understood. Here we have elucidated the stereochemical requirements for binding and activation by inositol phosphates, demonstrating that activation requires three adjacent phosphate groups and that other positions on the inositol ring can tolerate bulky substituents. We also demonstrate that there is allosteric communication between the inositol-binding site and the active site. The crystal structure of the HDAC1:MTA1 complex bound to a novel peptide-based inhibitor and to inositol hexaphosphate suggests a molecular basis of substrate recognition, and an entropically driven allosteric mechanism of activation.

The NuRD complex is a multi-protein transcriptional corepressor that couples histone deacetylase and ATP-dependent chromatin remodelling activities. The complex regulates the higher-order structure of chromatin, and has important roles in the regulation of gene expression, DNA damage repair and cell differentiation. HDACs 1 and 2 are recruited by the MTA1 corepressor to form the catalytic core of the complex. The histone chaperone protein RBBP4, has previously been shown to bind to the carboxy-terminal tail of MTA1. We show that MTA1 recruits a second copy of RBBP4. The crystal structure reveals an extensive interface between MTA1 and RBBP4. An EM structure, supported by SAXS and crosslinking, reveals the architecture of the dimeric HDAC1:MTA1:RBBP4 assembly which forms the core of the NuRD complex. We find evidence that in this complex RBBP4 mediates interaction with histone H3 tails, but not histone H4, suggesting a mechanism for recruitment of the NuRD complex to chromatin.

The death-inducing signaling complex (DISC) initiates death receptor-induced apoptosis. DISC assembly and activation are controlled by c-FLIP isoforms, which function as pro-apoptotic (c-FLIPL only) or anti-apoptotic (c-FLIPL/c-FLIPS) regulators of procaspase-8 activation. Current models assume that c-FLIP directly competes with procaspase-8 for recruitment to FADD. Using a functional reconstituted DISC, structure-guided mutagenesis, and quantitative LC-MS/MS, we show that c-FLIPL/S binding to the DISC is instead a co-operative procaspase-8-dependent process. FADD initially recruits procaspase-8, which in turn recruits and heterodimerizes with c-FLIPL/S via a hierarchical binding mechanism. Procaspase-8 activation is regulated by the ratio of unbound c-FLIPL/S to procaspase-8, which determines composition of the procaspase-8:c-FLIPL/S heterodimer. Thus, procaspase-8:c-FLIPL exhibits localized enzymatic activity and is preferentially an activator, promoting DED-mediated procaspase-8 oligomer assembly, whereas procaspase-8:c-FLIPS lacks activity and potently blocks procaspase-8 activation. This co-operative hierarchical binding model explains the dual role of c-FLIPL and crucially defines how c-FLIP isoforms differentially control cell fate., (Copyright © 2016 The Authors. Published by Elsevier Inc. All rights reserved.)

Histone deacetylase 3 (HDAC3), a member of the Class I subfamily of HDACs, is found in both the nucleus and the cytoplasm. Its roles in the nucleus have been well characterized, but its cytoplasmic roles are still not elucidated fully. We found that blocking HDAC3 activity using MI192, a compound specific for HDAC3, modulated tubulin acetylation in the human prostate cancer cell line PC3. A brief 1 h treatment of PC3 cells with MI192 significantly increased levels of tubulin acetylation and ablated the dynamic behaviour of microtubules in live cells. siRNA-mediated knockdown (KD) of HDAC3 in PC3 cells, significantly increased levels of tubulin acetylation, and overexpression reduced it. However, the active HDAC3-silencing mediator of retinoic and thyroid receptors (SMRT)-deacetylase-activating domain (DAD) complex did not directly deacetylate tubulin in vitro. These data suggest that HDAC3 indirectly modulates tubulin acetylation., (© 2015 Authors.)

Class IIa histone deacetylases repress transcription of target genes. However, their mechanism of action is poorly understood because they exhibit very low levels of deacetylase activity. The class IIa HDACs are associated with the SMRT/NCoR repression complexes and this may, at least in part, account for their repressive activity. However, the molecular mechanism of recruitment to co-repressor proteins has yet to be established. Here we show that a repeated peptide motif present in both SMRT and NCoR is sufficient to mediate specific interaction, with micromolar affinity, with all the class IIa HDACs (HDACs 4, 5, 7, and 9). Mutations in the consensus motif abrogate binding. Mutational analysis of HDAC4 suggests that the peptide interacts in the vicinity of the active site of the enzyme and requires the "closed" conformation of the zinc-binding loop on the surface of the enzyme. Together these findings represent the first insights into the molecular mechanism of recruitment of class IIa HDACs to the SMRT/NCoR repression complexes., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Recent proteomic studies have identified a novel histone deacetylase complex that is upregulated during mitosis and is associated with cyclin A. This complex is conserved from nematodes to man and contains histone deacetylases 1 and 2, the MIDEAS corepressor protein and a protein called DNTTIP1 whose function was hitherto poorly understood. Here, we report the structures of two domains from DNTTIP1. The amino-terminal region forms a tight dimerization domain with a novel structural fold that interacts with and mediates assembly of the HDAC1:MIDEAS complex. The carboxy-terminal domain of DNTTIP1 has a structure related to the SKI/SNO/DAC domain, despite lacking obvious sequence homology. We show that this domain in DNTTIP1 mediates interaction with both DNA and nucleosomes. Thus, DNTTIP1 acts as a dimeric chromatin binding module in the HDAC1:MIDEAS corepressor complex., (© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Gene expression is controlled through the recruitment of large coregulator complexes to specific gene loci to regulate chromatin structure by modifying epigenetic marks on DNA and histones. Metastasis-associated protein 1 (MTA1) is an essential component of the nucleosome remodelling and deacetylase (NuRD) complex that acts as a scaffold protein to assemble enzymatic activity and nucleosome targeting proteins. MTA1 consists of four characterised domains, a number of interaction motifs, and regions that are predicted to be intrinsically disordered. The ELM2-SANT domain is one of the best-characterised regions of MTA1, which recruits histone deacetylase 1 (HDAC1) and activates the enzyme in the presence of inositol phosphate. MTA1 is highly upregulated in several types of aggressive tumours and is therefore a possible target for cancer therapy. In this review, we summarise the structure and function of the four domains of MTA1 and discuss the possible functions of less well-characterised regions of the protein.

β-Lactamases inactivate β-lactam antibiotics by hydrolysis of their endocyclic β-lactam bond and are a major cause of antibiotic resistance in pathogenic bacteria. The zinc dependent metallo-β-lactamase enzymes are of particular concern since they are located on highly transmissible plasmids and have a broad spectrum of activity against almost all β-lactam antibiotics. We present here essentially complete (>96%) backbone and sidechain sequence-specific NMR resonance assignments for the Bacillus cereus subclass B1 metallo-β-lactamase, BcII, and for its complex with R-thiomandelic acid, a broad spectrum inhibitor of metallo-β-lactamases. These assignments have been used as the basis for determination of the solution structures of the enzyme and its inhibitor complex and can also be used in a rapid screen for other metallo-β-lactamase inhibitors.

STAR (signal transduction and activation of RNA) proteins are a family of RNA-binding proteins that regulate post-transcriptional gene regulation events at various levels, such as pre-mRNA alternative splicing, RNA export, translation and stability. Most of these proteins are regulated by signalling pathways through post-translational modifications, such as phosphorylation and arginine methylation. These proteins share a highly conserved RNA-binding domain, denoted STAR domain. Structural investigations of this STAR domain in complex with RNA have highlighted how a subset of STAR proteins specifically recognizes its RNA targets. The present review focuses on the structural basis of RNA recognition by this family of proteins.

In the past few years, RNA molecules have been revealed to be at the center of numerous biological processes. Long considered as passive molecules transferring genetic information from DNA to proteins, it is now well established that RNA molecules play important regulatory roles. Associated with that, the number of identified RNA binding proteins (RBPs) has increased considerably and mutations in RNA molecules or RBP have been shown to cause various diseases, such as cancers. It is therefore crucial to understand at the molecular level how these proteins specifically recognise their RNA targets in order to design new generation drug therapies targeting protein-RNA complexes. Nuclear magnetic resonance (NMR) is a particularly well-suited technique to study such protein-RNA complexes at the atomic level and can provide valuable information for new drug discovery programs. In this article, we describe the NMR strategy that we and other laboratories use for screening optimal conditions necessary for structural studies of protein-single stranded RNA complexes, using two proteins, Sam68 and T-STAR, as examples., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Metallo-β-lactamases, enzymes which inactivate β-lactam antibiotics, are of increasing biological and clinical significance as a source of antibiotic resistance in pathogenic bacteria. In the present study we describe the high-resolution solution NMR structures of the Bacillus cereus metallo-β-lactamase BcII and of its complex with R-thiomandelic acid, a broad-spectrum inhibitor of metallo-β-lactamases. This is the first reported solution structure of any metallo-β-lactamase. There are differences between the solution structure of the free enzyme and previously reported crystal structures in the loops flanking the active site, which are important for substrate and inhibitor binding and catalysis. The binding of R-thiomandelic acid and the roles of active-site residues are defined in detail. Changes in the enzyme structure upon inhibitor binding clarify the role of the mobile β3-β4 loop. Comparisons with other metallo-β-lactamases highlight the roles of individual amino-acid residues in the active site and the β3-β4 loop in inhibitor binding and provide information on the basis of structure-activity relationships among metallo-β-lactamase inhibitors.

Nuclear receptors are transcription factors that regulate gene expression through the ligand-controlled recruitment of a diverse group of proteins known as coregulators. Most nuclear receptor coregulators function in large multi-protein complexes that modify chromatin and thereby regulate the transcription of target genes. Structural and functional studies are beginning to reveal how these complexes are assembled bringing together multiple functionalities that mediate: recruitment to specific genomic loci through interaction with transcription factors; recruitment of enzymatic activities that either modify or remodel chromatin and targeting the complexes to their chromatin substrate. These activities are regulated by post-translational modifications, alternative splicing and small signalling molecules. This review focuses on our current understanding of coregulator complexes and aims to highlight the common principles that are beginning to emerge.

Class I histone deacetylases (HDAC1, HDAC2, and HDAC3) are recruited by cognate corepressor proteins into specific transcriptional repression complexes that target HDAC activity to chromatin resulting in chromatin condensation and transcriptional silencing. We previously reported the structure of HDAC3 in complex with the SMRT corepressor. This structure revealed the presence of inositol-tetraphosphate [Ins(1,4,5,6)P4] at the interface of the two proteins. It was previously unclear whether the role of Ins(1,4,5,6)P4 is to act as a structural cofactor or a regulator of HDAC3 activity. Here we report the structure of HDAC1 in complex with MTA1 from the NuRD complex. The ELM2-SANT domains from MTA1 wrap completely around HDAC1 occupying both sides of the active site such that the adjacent BAH domain is ideally positioned to recruit nucleosomes to the active site of the enzyme. Functional assays of both the HDAC1 and HDAC3 complexes reveal that Ins(1,4,5,6)P4 is a bona fide conserved regulator of class I HDAC complexes., (Copyright © 2013 The Authors. Published by Elsevier Inc. All rights reserved.)

Co-repressor proteins, such as SMRT and NCoR, mediate the repressive activity of unliganded nuclear receptors and other transcription factors. They appear to act as intrinsically disordered "hub proteins" that integrate the activities of a range of transcription factors with a number of histone modifying enzymes. Although these co-repressor proteins are challenging targets for structural studies due to their largely unstructured character, a number of structures have recently been determined of co-repressor interaction regions in complex with their interacting partners. These have yielded considerable insight into the mechanism of assembly of these complexes, the structural basis for the specificity of the interactions and also open opportunities for targeting these interactions therapeutically., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

Histone deacetylase enzymes (HDACs) are emerging cancer drug targets. They regulate gene expression by removing acetyl groups from lysine residues in histone tails, resulting in chromatin condensation. The enzymatic activity of most class I HDACs requires recruitment into multi-subunit co-repressor complexes, which are in turn recruited to chromatin by repressive transcription factors. Here we report the structure of a complex between an HDAC and a co-repressor, namely, human HDAC3 with the deacetylase activation domain (DAD) from the human SMRT co-repressor (also known as NCOR2). The structure reveals two remarkable features. First, the SMRT-DAD undergoes a large structural rearrangement on forming the complex. Second, there is an essential inositol tetraphosphate molecule--D-myo-inositol-(1,4,5,6)-tetrakisphosphate (Ins(1,4,5,6)P(4))--acting as an 'intermolecular glue' between the two proteins. Assembly of the complex is clearly dependent on the Ins(1,4,5,6)P(4), which may act as a regulator--potentially explaining why inositol phosphates and their kinases have been found to act as transcriptional regulators. This mechanism for the activation of HDAC3 appears to be conserved in class I HDACs from yeast to humans, and opens the way to novel therapeutic opportunities.

The potential of flavocytochrome P450 BM3 (CYP102A1) from Bacillus megaterium for biocatalysis and biotechnological application is widely acknowledged. The catalytic and structural analysis of the Ala82Phe mutant of P450 BM3 has shown that filling a hydrophobic pocket near the active site improved the binding of small molecules, such as indole (see Huang et al., J. Mol. Biol., 2007, 373, 633) and styrene. In this paper, additional mutations at Thr438 are shown to decrease the binding of and catalytic activity towards laurate, whereas they significantly increased the stereo-specificity of styrene epoxidation. Production of R-styrene oxide with 48% and 64% e.e., respectively, was achieved by the Ala82Phe-Thr438Leu and Ala82Phe-Thr438Phe mutants. These structure-based mutants of P450 BM3 illustrate the promise of rational design of synthetically useful biocatalysts for regio- and stereo- specific mono-oxygenation reactions.

Eukaryotic transcriptional repressors function by recruiting large coregulatory complexes that target histone deacetylase enzymes to gene promoters and enhancers. Transcriptional repression complexes, assembled by the corepressor NCoR and its homolog SMRT, are crucial in many processes, including development and metabolic physiology. The core repression complex involves the recruitment of three proteins, HDAC3, GPS2 and TBL1, to a highly conserved repression domain within SMRT and NCoR. We have used structural and functional approaches to gain insight into the architecture and biological role of this complex. We report the crystal structure of the tetrameric oligomerization domain of TBL1, which interacts with both SMRT and GPS2, and the NMR structure of the interface complex between GPS2 and SMRT. These structures, together with computational docking, mutagenesis and functional assays, reveal the assembly mechanism and stoichiometry of the corepressor complex.

We have incorporated CYP3A4 (cytochrome P450 3A4) and CPR (NADPH-cytochrome P450 reductase) into liposomes with a high lipid/protein ratio by an improved method. In the purified proteoliposomes, CYP3A4 binds testosterone with Kd (app)=36±6 μM and Hill coefficient=1.5±0.3, and 75±4% of the CYP3A4 can be reduced by NADPH in the presence of testosterone. Transfer of the first electron from CPR to CYP3A4 was measured by stopped-flow, trapping the reduced CYP3A4 as its Fe(II)-CO complex and measuring the characteristic absorbance change. Rapid electron transfer is observed in the presence of testosterone, with the fast phase, representing 90% of the total absorbance change, having a rate of 14±2 s(-1). Measurements of the first electron transfer were performed at various molar ratios of CPR/CYP3A4 in proteoliposomes; the rate was unaffected, consistent with a model in which first electron transfer takes place within a relatively stable CPR-CYP3A4 complex. Steady-state rates of NADPH oxidation and of 6β-hydroxytestosterone formation were also measured as a function of the molar ratio of CPR/CYP3A4 in the proteoliposomes. These rates increased with increasing CPR/CYP3A4 ratio, showing a hyperbolic dependency indicating a Kd (app) of ~0.4 μM. This suggests that the CPR-CYP3A4 complex can dissociate and reform between the first and second electron transfers.

The nuclear receptor peroxisome proliferator-activated receptor-gamma (PPARgamma) has important roles in adipogenesis and immune response as well as roles in both lipid and carbohydrate metabolism. Although synthetic agonists for PPARgamma are widely used as insulin sensitizers, the identity of the natural ligand(s) for PPARgamma is still not clear. Suggested natural ligands include 15-deoxy-delta12,14-prostaglandin J2 and oxidized fatty acids such as 9-HODE and 13-HODE. Crystal structures of PPARgamma have revealed the mode of recognition for synthetic compounds. Here we report structures of PPARgamma bound to oxidized fatty acids that are likely to be natural ligands for this receptor. These structures reveal that the receptor can (i) simultaneously bind two fatty acids and (ii) couple covalently with conjugated oxo fatty acids. Thermal stability and gene expression analyses suggest that such covalent ligands are particularly effective activators of PPARgamma and thus may serve as potent and biologically relevant ligands.

Cytochrome P450BM3 (CYP102A1) from Bacillus megaterium, a fatty acid hydroxylase, is a member of a very large superfamily of monooxygenase enzymes. The available crystal structures of the enzyme show non-productive binding of substrates with their omega-end distant from the iron in a hydrophobic pocket at one side of the active site. We have constructed and characterised mutants in which this pocket is filled by large hydrophobic side-chains replacing alanine at position 82. The mutants having phenylalanine or tryptophan at this position have very much (approximately 800-fold) greater affinity for substrate, with a greater conversion of the haem iron to the high-spin state, and similarly increased catalytic efficiency. The enzyme as isolated contains bound palmitate, reflecting this much higher affinity. We have determined the crystal structure of the haem domain of the Ala82Phe mutant with bound palmitate; this shows that the substrate is binding differently from the wild-type enzyme but still distant from the haem iron. Detailed analysis of the structure indicates that the tighter binding in the mutant reflects a shift in the conformational equilibrium of the substrate-free enzyme towards the conformation seen in the substrate complex rather than differences in the enzyme-substrate interactions. On this basis, we outline a sequence of events for the initial stages of the catalytic cycle. The Ala82Phe and Ala82Trp mutants are also very much more effective catalysts of indole hydroxylation than the wild-type enzyme, suggesting that they will be valuable starting points for the design of mutants to catalyse synthetically useful hydroxylation reactions.