The Structure of Monoamine Oxidase from Aspergillus niger Provides a Molecular Context for Improvements in Activity Obtained by Directed Evolution

Atkin, Kate E.; Reiss, Renate; Koehler,; Bailey, Kevin; Hart, Sam; Turkenburg, J.P.; Turner, Nicholas J.; Brzozowski, A.M.; Grogan, Gideon

doi:10.1016/j.jmb.2008.09.090

Cited by 76 publications

(75 citation statements)

References 41 publications

(50 reference statements)

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“…Fractions containing XplA-heme were pooled, concentrated, and then applied to a Superdex TM 200 column (Amersham Biosciences) gel-filtration column (equilibrated in 50 mM phosphate buffer, pH 8, containing 300 mM sodium chloride) to yield protein for crystallization and solution assays. Before crystallization, the hexahistidine tag was cleaved from the XplA heme domain using protease 3C (Novagen) using a procedure described previously (15).…”

Section: Methodsmentioning

confidence: 99%

“…Mutation of Met-318 however resulted in a decreased affinity for RDX and a 20-fold decrease in k cat /K m overall. Mutations to Met-220 in the access channel of 14␣-sterol demethylase (Cyp51A) of Aspergillus fumigatus have been previously shown to disrupt the binding of itraconazole to that enzyme, conferring drug resistance to the host mutant organisms (44), and, conversely, mutation of an isoleucine residue to methionine in the access channel of the amine oxidase from Aspergillus niger has been shown to increase the rate of turnover, perhaps as a result of improved access to the active site (15). In addition to flexibility, methionine clusters have also been observed to exert electronic effects through their capacity for oxidation to methionine sulfoxide, for example in the potassium channel Slo1 (45), where oxidation of methionines within the channel was thought to enhance the activity of that channel when under oxidative stress, but there is no structural evidence for the oxidation of methionines 318, 322, or 394 in any structures of XplA-heme, that might suggest a role in oxygen sensitivity in this enzyme.…”

Section: Structure Of Xpla-hemementioning

confidence: 99%

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The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

Sabbadin

Jackson

Haider

et al. 2009

Journal of Biological Chemistry

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XplA is a cytochrome P450 of unique structural organization, consisting of a heme-domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2 1 to a resolution of 1.9 Å and as a monomer in space group P2 1 2 1 2 to a resolution of 1.5 Å , with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogenbonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 3 Ala and Ala-395 3 Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors. Royal demolition explosive (RDX)2 or cyclotrimethylenetrinitramine 1 (see Fig. 1) is a widely used explosive compound with both military and civil applications. The extensive global usage of RDX has resulted in concerns over environmental contamination, because it is both recalcitrant to degradation, leading to contamination in soil and ground water, and a potent convulsant and possible carcinogen. The bioremediation of RDX has thus been the focus of increasing research in recent years, with a number of bacterial strains reported to catalyze its degradation (1-3). Among these, Bruce and co-workers, using a selective enrichment technique, isolated Rhodococcus rhodochrous strain 11Y from an RDX-contaminated site, which was able to grow on RDX as the sole nitrogen source (4). The products of biotransformation of RDX by this bacterium were shown to be nitrite and formaldehyde. A gene cluster in strain 11Y essential for RDX degradation was identified and shown to contain a novel cytochrome P450, termed XplA, and a redox partner, XplB, which were shown together to be capable of catalyzing the biotransformation of RDX in vitro (5). Near identical xplA and xplB genes have now been identified in strains within the Actinomycetales isolated from geographically distinct sites (6). Interestingly, these genes have been found to be plasmid encoded providing compelling evidence for recent lateral gene transfer. In the intere...

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Section: Methodsmentioning

confidence: 99%

Section: Structure Of Xpla-hemementioning

confidence: 99%

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

Sabbadin

Jackson

Haider

et al. 2009

Journal of Biological Chemistry

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“…oxidase MAO N [32] is a soluble enzyme supposed to be located in the peroxisome and has an overall globular structure similar to that of PAOs (Fig. 3A).…”

Section: Lsd1 Is a Flavin-dependent Amine Oxidasementioning

confidence: 99%

“…Sequence identity values refer to residues belonging to the AOD of each enzyme. PAO, polyamine oxidase [30]; FMS1, yeast polyamine oxidase [31]; MAO, monoamine oxidase [32][33][34]; LAAO, L-amino acid oxidase [37]; GOX, glycine oxidase [48]; MSOX, monomeric sarcosine oxidase [49]. have been evolutionarily adapted to the enzyme function and type of substrate.…”

Section: Lsd1 Is a Flavin-dependent Amine Oxidasementioning

confidence: 99%

New roles of flavoproteins in molecular cell biology: Histone demethylase LSD1 and chromatin

et al. 2009

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Lysine‐specific demethylase 1 (LSD1) is an enzyme that removes methyl groups from mono‐ and dimethylated Lys4 of histone H3, a post‐translational modification associated with gene activation. Human LSD1 was the first histone demethylase to be discovered and this enzymatic activity is conserved among eukaryotes. LSD1 has been identified in a number of chromatin‐remodeling complexes that control gene transcription and its demethylase activity has also been linked to pathological processes including tumorigenesis. The 852‐residue sequence of LSD1 comprises an amine oxidase domain which identifies a family of enzymes that catalyze the FAD‐dependent oxidation of amine substrates ranging from amino acids to aromatic neurotransmitters. Among these proteins, LSD1 is peculiar in that it acts on a protein substrate in the nuclear environment of chromatin‐remodeling complexes. This functional divergence occurred during evolution from the eubacteria to eukaryotes by acquisition of additional domains such as the SWIRM domain. The N‐terminal part of LSD1, predicted to be disordered, contains linear motifs that might represent functional sites responsible for the association of this enzyme with a variety of transcriptional protein complexes. LSD1 shares structural features with other flavin amine oxidases, including the overall fold of the amine oxidase domain region and details in the active site that are relevant for amine substrate oxidation.

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“…It is not a trivial task to identify remote sites in an enzyme at which saturation mutagenesis can be expected to generate mutations that would trigger allosterically-induced domain movements with the creation of a structurally new binding pocket in the absence of an effector. Suggestions about movements of the NADP-binding domain believed to be coupled to the catalytic mechanism of PAMO (23) led us to consider mutations strategically located in regions distal from the active site that might induce such structural reorganizations (28)(29)(30). Close inspection of the PAMO X-ray structure shows that the number of likely possibilities is in fact limited.…”

Section: Resultsmentioning

confidence: 99%

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

Acevedo

Reetz

2010

Proc. Natl. Acad. Sci. U.S.A.

120

118

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The molecular basis of allosteric effects, known to be caused by an effector docking to an enzyme at a site distal from the binding pocket, has been studied recently by applying directed evolution. Here, we utilize laboratory evolution in a different way, namely to induce allostery by introducing appropriate distal mutations that cause domain movements with concomitant reshaping of the binding pocket in the absence of an effector. To test this concept, the thermostable Baeyer-Villiger monooxygenase, phenylacetone monooxygenase (PAMO), was chosen as the enzyme to be employed in asymmetric Baeyer-Villiger reactions of substrates that are not accepted by the wild type. By using the known X-ray structure of PAMO, a decision was made regarding an appropriate site at which saturation mutagenesis is most likely to generate mutants capable of inducing allostery without any effector compound being present. After screening only 400 transformants, a double mutant was discovered that catalyzes the asymmetric oxidative kinetic resolution of a set of structurally different 2-substituted cyclohexanone derivatives as well as the desymmetrization of three different 4-substituted cyclohexanones, all with high enantioselectivity. Molecular dynamics (MD) simulations and covariance maps unveiled the origin of increased substrate scope as being due to allostery. Large domain movements occur that expose and reshape the binding pocket. This type of focused library production, aimed at inducing significant allosteric effects, is a viable alternative to traditional approaches to "designed" directed evolution that address the binding site directly.allosteric effects | enzymes | molecular dynamics simulations | protein engineering P rotein allostery has been recognized as a positive or negative cooperative event, leading to a structural change at the binding site as a result of distal docking of a molecule acting as an effector (1-5). Allosteric effects can be influenced by such factors as variation in pH, temperature, ionic strength, and covalent modification as well as mutational changes. A variety of experimental and computational techniques have been utilized to unravel the intricacies of this phenomenon (1-5), but also to achieve useful applications such as the creation of protein switches (6). Among the techniques that have been applied to address these challenges is directed evolution, a method that is generally used to engineer the catalytic profiles of enzymes or the binding properties of proteins (7-11). In the endeavor to make directed evolution of enantioselective enzymes more efficient than in the past, we recently introduced the concept of iterative saturation mutagenesis according to which sites around the binding pocket are randomized iteratively, a given site being composed of one or more amino acid positions in the protein (12-14.) When applying this technique to enzymes displaying strong allosteric effects or coupled motions along the reaction coordinate (15), it is necessary to consider such phenomena to make reasonable...

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The Structure of Monoamine Oxidase from Aspergillus niger Provides a Molecular Context for Improvements in Activity Obtained by Directed Evolution

Cited by 76 publications

References 41 publications

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

The 1.5-Å Structure of XplA-heme, an Unusual Cytochrome P450 Heme Domain That Catalyzes Reductive Biotransformation of Royal Demolition Explosive

New roles of flavoproteins in molecular cell biology: Histone demethylase LSD1 and chromatin

Induced allostery in the directed evolution of an enantioselective Baeyer–Villiger monooxygenase

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