Dissertations / Theses on the topic 'CYP101C1'

Human drug metabolites are frequently biologically active, with many implications for human health. Pharmaceutical companies have become increasingly aware of the need to identify and test these metabolites. The P450 BM3 enzyme from Bacillus megaterium offers substantial advantages to the current methods of metabolite synthesis, as its soluble, catalytically self-sufficient nature, coupled with its high catalytic activity, make P450 BM3 ideal for engineering towards specificity for human drugs. The highly-active I401P BM3 mutant was characterized for its reactivity towards human drugs and for the development of a human P450-like metabolite profile. The I401P mutant exhibits binding to molecules including alkaloids, steroids, and azole drugs, along with many other compounds. I401P binds/oxidizes human CYP substrates, including alosetron, phenacetin, caffeine, nicotine and diclofenac. LC-MS product identification shows that I401P BM3 forms 4OH-diclofenac, the major human metabolite for diclofenac. I401P BM3 also produces nornicotine, the second major human metabolite of nicotine. I401P BM3 also forms theophylline, theobromine and paraxanthine, the three major human metabolites of caffeine. Thermostability (DSC) data show that the I401P mutation destabilizes the BM3 heme domain in both its substrate-free and substrate-bound forms. The I401P heme domain X-ray crystal structure reinforces previous structural observations that the Pro401 mutation causes the BM3 protein to adopt a high-spin, "substrate-bound" state, with a displaced heme iron axial water, producing a "catalytically primed" mutant with greater diversity in substrate selectivity. The destabilisation of the BM3 heme domain structure due to the Pro401 mutation increases conformational plasticity in this mutant, allowing it to function as a platform for future mutagenesis aimed at improved binding and metabolite yield from specific drug substrates. Further proline mutations (A330P, A330P/I401P and A82F/F87V/I401P) were examined for increased affinity for drug substrates. The A330P mutant shows no novel drug substrate specificity, despite its reported affinity for small molecules. The A330P/I401P double mutant demonstrates weak binding to WT BM3 and I401P substrates, but no synergistic effects were obtained by combining the two mutations. The double mutant exhibits very low solvent tolerance and significant structural destabilisation. DSC data confirms this, with the double mutant destabilising the BM3 heme domain by up to 20 °C. Initial work with the A82F/F87V/I401P mutant showed increased affinity for A82F/F87V- and I401P-type substrates, including diclofenac. LC-MS product analysis confirms that the A82F/F87V/I401P mutant oxidises diclofenac into its major human metabolite 4OH-diclofenac. These data indicate that human-like oxidation reactions are feasible with BM3 mutants. In this work, proline insertion mutants were generated that introduced novel affinity for biotechnologically relevant substrates. In particular the I401P mutant offers an excellent platform for future biotechnological engineering.

High catalytic activity and a broad substrate range are characteristic of P450 fusion enzymes of the CYP102A class. P450 BM3 (CYP102A1, BM3) is a paradigm for the P450 fusion enzymes and is accredited with the highest monooxygenase activity in the P450 superfamily, a property which has led to its engineering and exploitation for biotechnologically valuable oxidation reactions. Initial research in the thesis focused on characterisation of a novel P450-redox partner fusion enzyme from the thermophilic fungus Myceliophthora thermophila (CYP505A30, P450MT1). Sequence alignments revealed a P450 domain and a diflavin P450 reductase domain with high sequence similarity to BM3’s domains (41% and 31% amino acid identity, respectively). The purified 118 kDa protein is soluble and exhibits characteristic P450 spectral properties, giving a Soret absorption shift to 450 nm upon binding CO to its ferrous heme iron. Binding titrations of intact P450 MT1 and its expressed P450 (heme) domain with fatty acid substrates and imidazole-based inhibitors revealed type I (blue) and II (red) Soret shifts, respectively, typical of other members of the P450 superfamily, and enabled determination of substrate binding constants. HPLC analysis confirmed stoichiometric amounts of bound FAD and FMN cofactors. Subsequent kinetic and biochemical studies included stopped-flow kinetic experiments showing that NADPH-dependent reduction of P450 MT1’s FAD cofactor occurs with a rate constant of ~150 s-1 at 20 °C. P450 MT1 has an unconventional substrate hydroxylation profile for saturated fatty acids. It hydroxylates these substrates predominantly at positions ω-1, ω-2 and ω-3. However, an unusual property of this enzyme is observed in its strong preference (~85% of total converted product) for either the ω-1 or the ω-2 position on odd and even chain length fatty acids, respectively. However, it displays higher selectivity for branched chain fatty acids over straight chain fatty acids, e.g. for the substrate iso-myristic acid, similar to BM3’s properties. Other work done focused on biophysical characterisation of the model P450-reductase fusion enzyme P450 BM3 from Bacillus megaterium. A combination of alternative structural techniques to X-ray crystallography were used to characterise the enzyme. More specifically, electron microscopy (EM) and nuclear magnetic resonance (NMR) were used to gain greater insights into the intimate associations of the enzyme monomers in BM3’s dimeric structure. These studies led to the first structural insights into how P450 BM3’s dimeric complex is organised. Dimerisation in BM3 arises predominantly from self-association of the enzyme’s FAD domains, and wild-type and mutant BM3 FAD domain forms were also characterised. Key FAD domain mutations that prevented intra-/inter-monomer disulphide bond formation facilitated the crystallization and determination of the FAD domain structure, the final part of the BM3 enzyme to have its three dimensional structure resolved. Data reported in this thesis give new insights into the biochemistry of biotechnologically important P450 monooxygenase enzymes from mesophilic and thermophilic microorganisms.

Le cytochrome p450 CYP102A1, mieux connu sous le nom de BM3, provient de la bactérie Bacillus megaterium. Cette enzyme possède un groupement prosthétique hémique lui permettant de catalyser l’insertion d’oxygène dans un lien carbone-hydrogène menant généralement à une hydroxylation du substrat, ce qui en fait une monooxygénase. Ce genre de réaction demeure jusqu’à aujourd’hui difficile à effectuer par chimie traditionnelle ce qui confère un intérêt particulier à cette enzyme. Au contraire des autres cytochromes p450, BM3 est soluble (et non membranaire) et est naturellement fusionnée à son partenaire réductase formant ainsi une seule chaîne polypeptidique. Ainsi, au cours des dernières années, BM3 a attiré beaucoup d’attention de la part de l’industrie de la chimie fine et pharmaceutique due à son potentiel biocatalytique important. Cependant, son usage en industrie est restreint par son instabilité ainsi que par le coût prohibitif du cofacteur qui lui est nécessaire pour catalyser ses réactions, le NADPH. Cette thèse décrit le développement de différentes stratégies visant à libérer les réactions effectuées avec BM3 de leur dépendance au NADPH, tout en maximisant le rendement spécifique de la monooxygénase. En place du NADPH, deux autres cofacteurs de moindre coût furent utilisés comme alternative, soit le NADH et le N-benzyle-1,4- dihydronicotinamide (NBAH) en utilisant le mutant R966D/W1046S de BM3. Afin de maximiser le rendement spécifique de BM3, l’une des stratégies de cette thèse, l’optimisation du milieu réactionnel, repose sur deux éléments clés, soit favoriser la stabilité du cofacteur, car celui-ci est plus instable que l’enzyme elle-même, ainsi que d’abaisser au minimum la température de la réaction, car nous avons constaté que ceci avait pour effet d’augmenter le couplage entre les réactions réductase et monooxygénase et donc la stabilité de l’enzyme. L’effet net de la réaction ainsi optimisée fut d’augmenter le rendement spécifique du mutant R966D/W1046S par un facteur situé entre 2 et 2.6 en fonction du cofacteur utilisé. D’autre part, deux stratégies d’ingénierie enzymatique furent explorées afin de générer des mutations pouvant augmenter la performance de BM3. L’une d’entre elles, la mutagenèse par consensus guidé, généra une librairie de mutants de laquelle les mutants NTD5 et NTD6 furent identifiés, augmentant le rendement spécifique de l’enzyme comparativement à leur parent, R966D/W1046S, par un facteur de 5.2 et 2.3 pour le NBAH et le NADH, respectivement. L’autre stratégie explorée fut d’appliquer une pression sélective sur la bactérie Bacillus megaterium pour forcer, par évolution expérimentale, la performance de l’enzyme. De cette stratégie, un nouveau mutant de BM3 nommé DE, possédant 34 acides aminés substitués sur sa séquence, fut généré. Ce dernier a démontré une plus forte résistance aux solvants organiques ainsi qu’une augmentation de son rendement spécifique vis-à-vis le NADPH et le NADH d’un facteur de 1.23 et 1.76, comparativement à BM3 sauvage, respectivement. Les stratégies décrites dans cette thèse présentent une amélioration significative du rendement spécifique de BM3 ainsi que deux iii nouvelles méthodologies avec lesquelles une enzyme peut être optimisée et de nouvelles mutations bénéfiques identifiées.
The p450 cytochrome CYP102A1, better known as BM3, comes from the bacteria Bacillus megaterium. This enzyme possesses a prosthetic heme group enabling it to catalyze the insertion of oxygen into a carbon-hydrogen bond generally resulting in the hydroxylation of the substrate, the enzyme is therefore a monooxygenase. This type of reaction remains difficult to achieve by traditional chemistry. Unlike other p450 cytochromes, BM3 is soluble (is not membrane bound) and is naturally fused to its reductase partner forming a single polypeptide chain. As such, in recent years, BM3 has garnered much attention from the pharmaceutical and fine chemical industries, due to its high biocatalytic potential. However, its use in industry remains constrained by its instability as well as by the prohibitive cost of its cofactor, NADPH. This thesis describes the development of different strategies aiming at liberating reactions driven with BM3 from their dependence to NADPH whilst maximizing the specific yield of the monooxygenase. Instead of NADPH, two other inexpensive cofactors were used, namely NADH and N-benzyl-1,4-dihydronicotinamide (NBAH) by using the BM3 mutant R966D/W1046S. To maximize BM3 specific yield, one of the strategies used in this thesis work, the optimization of the reaction medium, rested on two key elements. Firstly, favouring the stabilization of the cofactor, as it was found to be more unstable than the enzyme itself and secondly lowering the reaction temperature as this effectively augmented oxidase/reductase reactions coupling and as such the stability of the enzyme. The net effect of the optimized reaction was to enhance the specific yield of the BM3 mutant R966D/W1046S by a factor of 2 and 2,6 depending on which cofactor was used. Two other enzymatic engineering strategies were explored to generate mutations which could enhance the performance of BM3. One of these, consensus guided mutagenesis, generated a library of mutants from which mutants NTD5 and NTD6 were identified enhancing the specific yield of the enzyme comparatively to their parent, R966D/W1046S, by a factor of 5,24 and 2,3 for NBAH and NADH respectively. The other strategy explored was to apply a selective pressure on Bacillus megaterium to force, by experimental evolution, the performance of the enzyme. From this strategy, a new mutant of BM3 called DE, possessing 34 new amino acid substitutions, was generated. This new mutant displayed a greater resistance to organic solvents as well as an augmentation of specific yields when used alongside NADPH and NADH comparatively to wild type BM3 by a factor of 1,23 and 1,76 respectively. The strategies described in this thesis allowed a significative enhancement of BM3 specific yield as well as represent two new methodologies by which new beneficial mutations can be identified.

The cytochrome P450 enzymes CYP101B1 and CYP101C1 from Novosphingobium aromaticivorans DSM12444 are homologues of the CYP101D1 and CYP101D2 enzymes from the same bacterium and CYP101A1 (P450cam) from Pseudomonas putida. Both enzymes can efficiently hydroxylate norisoprenoids and related substrates in combination with the same ferredoxin reductase, ArR and a [2Fe-2S] ferredoxin, Arx, electron transfer partners. Even though the physiological substrates for both the enzymes are yet to be confirmed, the crystal structure of CYP101C1 bound to β -ionone and modelled structure of CYP101B1 has been generated. The Met82 residue of CYP101C1 aligns with the His85 residue of CYP101B1. In the crystallographic structure, this Met82 residue of CYP101C1, interacts with the carbonyl group of β -ionone, which makes it an interesting site for mutation as these could potentially alter the activity and hydroxylation of norisoprenoid substrates. CYP101B1 oxidised ẞ -ionone with the highest product formation rate (1010 ± 60 min-1). The CYP101C1 enzyme oxidised β -ionol with the highest product formation rate (1130 ± 30 min-1), whereas, the M82L-CYP101C1 mutant enzyme had the highest product formation rate (790 ± 22 min-1) with α-ionone. The selectivity for hydroxylation of norisoprenoids varies between CYP101B1 and CYP101C1. The M82L mutation however, did not change the selectivity for CYP101C1. For example, both β -damascone and β -ionone were hydroxylated at the C4 position by CYP101C1 and the M82L-CYP101C1 mutant. The CYP101B1 enzyme displayed an altered selectivity and hydroxylated these substrates predominantly at C3 position. When the substrate functional group was changed from a carbonyl to an alcohol (i.e. β-ionol), the hydroxylation occurred preferentially at the C3 position with all three enzymes. By comparing the oxidation of α -, β - and δ - substituted damascones, we found that the alkene moiety present inside the cyclohexyl ring did have an effect on the selectivity of oxidation. The β - substituted substrates are oxidised only at the C3 position by all three enzymes. The β - substituted substrates are oxidised at C3 position by CYP101B1 and at C4 position by CYP101C1 and M82L-CYP101C1. The δ - substituted substrate generates the 3,4-epoxide as the major product. To further explore the substrate range of CYP101B1 and CYP101C1, various substrates including cyclic ketones and cyclic esters were assessed to see if they induce enzyme activity and binding to the enzyme. The combinations of the best enzyme / substrates were then chosen to generate the oxidation metabolites in a larger quantity using whole-cell oxidation system to enable characterisation. The oxidation of 1-decalone by CYP101B1 generated a single major metabolite along with two minor products. The major product was characterized as 6-hydroxy-1-decalone and the minor product as 7-hydroxy-1-decalone. Comparison of the 1-decalone substrate to damascones, highlight the relationship of the oxidation metabolites 6-hydroxy-1-decalone to 4-hydroxy- β -damascone and 7-hydroxy-1-decalone to 3-hydroxy- β -damascone. Oxacyclotridecan-2-one is oxidised by CYP101B1 on a carbon opposite to the carbonyl group. Along with these, muscone and cyclopentadecanone show a dissociation constant similar to β -ionone with CYP101B1. However, the spin-state shift and activity induced by both of these substrates to CYP101B1 are comparatively smaller than ẞ -ionone. p-Tolyl acetate induced a large type-I spin-state shift and a weak binding to CYP101B1. It was oxidised at the benzylic methyl group, generating 7-hydroxy-p-tolyl acetate. Similarly, dihydroactinidiolide was also oxidised at the carbon opposite to the ester group generating 6-hydroxy-dihydroactinidiolide. However, this substrate induces a very small spin-state shift and was oxidised with low activity by CYP101B1. None of the tested substrate showed a spin-state shift larger than 10% HS or increased activity with CYP101C1.
Thesis (MPhil) -- University of Adelaide, School of Physical Sciences, 2020

The cytochrome P450 enzymes CYP101B1 and CYP101C1, which are from the aromatic hydrocarbon degrading bacterium Novosphingobium aromaticivorans DSM12444, can hydroxylate norisoprenoids with high activity and selectivity. With the aim of further understanding their substrate range, a selection of cyclic alkanes, ketones and alcohols were studied. Cycloalkanes were oxidised, but both enzymes displayed low binding affinity and productive activity. The presence of a ketone moiety in the cycloalkane skeleton significantly improved the substrate binding affinity and the oxidation activity. CYP101C1 catalysed the oxidation of the cycloalkanones at the C-2 position with high regioselectivity. The regioselectivity of CYP101B1 was different. It oxidised cycloalkanones at positions remote from the carbonyl group. This indicated that the binding orientation of the cyclic ketones in the active site of each enzyme must be different. Cyclic alcohols and cyclohexylacetic acid showed little to no activity with either enzyme. The introduction of an ester protecting group to these substrates significantly enhanced the monooxygenase activity. These substrates were oxidised regioselectively on the opposite side of the ring system to the ester directing group. For example, both enzymes preferentially oxidised the C-H bond at the C4, C5 and C7 position of the cyclohexyl, cyclooctyl and cyclododecyl ester compounds, respectively. In addition, certain linear ketones and esters were also found to be suitable substrates for these biocatalysts. CYP101B1 mediated metabolism of the tricyclic compounds adamantane, 1‐ and 2‐ adamantanol and 2‐adamantanone proceeds with low oxidation activity and multiple metabolites were identified. Insertion of a directing group (acetate/isobutyrate) at the alcohol of these adamantanols significantly increased the affinity, activity and coupling efficiency (productive use of reducing equivalents) of CYP101B1 compared to the parent compounds. This substrate engineering approach with these adamantyl derivatives led to a 65 to 122-fold higher product formation activity. The turnovers were also regioselective and in some instances stereoselective. Additionally, the amide N‐(1‐adamantyl)acetamide was oxidised efficiently by CYP101B1, whereas 1‐adamantylamine was not. Whole-cell biotransformation systems were used to generate the metabolites in good yield (g/L scale). Overall, the use of ester directing groups and the modification of the amine to an amide enabled CYP101B1 to oxidise the adamantane skeleton more efficiently and selectively. Wild-type (WT) CYP101B1 can catalyse the oxidation of aromatic substrates such as alkylbenzenes, alkylnaphthalenes and acenaphthene, but the binding affinities and the oxidation activities were low. Both the binding affinity and product formation activity of this enzyme for these hydrophobic substrates were enhanced using site-directed mutagenesis. The Histidine 85 (H85) of CYP101B1 aligns with tyrosine 96 of CYP101A1 (P450cam), which, in the latter enzyme forms the only hydrophilic interaction with its natural substrate, camphor. The H85 residue of CYP101B1 was therefore replaced with phenylalanine (F), and this H85F variant exhibited greater affinity and activity towards hydrophobic substrates. For instance, the product formation activity of the H85F variant for acenaphthene oxidation was increased sixfold to 245 nmol.nmol-CYP–1.min–1. This indicated that this residue is in the substrate binding pocket or the access channel of the enzyme. Methylcubanes have been used as mechanistic probes to differentiate between radical and cationic pathways in cytochrome P450 oxidation. A series of methylcubanes were designed which would place the methyl group close to reactive heme iron centre of CYP101B1. CYP101B1 efficiently oxidised the substituted methylcubane derivatives yielding the equivalent cubylmethanol in 93 ± 7 % yield. The cube was found to be intact in all the turnover products, and no methylcubanols or any other rearranged metabolites containing homocubyl were detected. These results were consistent with a rapid radical rebound step in these oxidations and argued against the involvement of any carbocation-based intermediates during the oxidation. The CYP101B1 system, which also combines a FAD-containing ferredoxin reductase and a [2Fe-2S] ferredoxin, was investigated with oxygenated aromatics including naphthols, naphthoquinones, dihydroxynaphthalene and phenols. In vitro NADH oxidation rates in both the presence and absence of CYP101B1 were fast with these substrates (≥800 min-1). Minimal metabolite formation was detected, and the majority of reducing equivalents were transformed into hydrogen peroxide. Large amount of H2O2 in these reactions in the absence of P450 indicated that the ferredoxin (Arx) and ferredoxin reductase (ArR) catalysed futile redox cycling with naphthoquinones giving rise to the uncoupling of the reducing equivalents. Further examination of naphthols and naphthoquinones together with 2-adamantyl acetate in the fully reconstituted CYP101B1 turnovers demonstrated that the presence of naphthoquinones led to diminished product formation as they interfere with the electron transfer process. This type of uncoupling in the bacterial P450 electron transfer partners containing ferredoxin system would be considered an additional form of uncoupling over those which arise in the P450 active site.
Thesis (Ph.D.) -- University of Adelaide, School of Physical Sciences, 2019

Cette thèse comporte quatre fichiers vidéo. This thesis comes with four video files.
L'industrie chimique mondiale est en pleine mutation, cherchant des solutions pour rendre la synthèse organique classique plus durable. Une telle solution consiste à passer de la catalyse chimique classique à la biocatalyse. Bien que les avantages des enzymes incluent leur stéréo, régio et chimiosélectivité, cette sélectivité réduit souvent leur promiscuité. Les efforts requis pour adapter la fonction enzymatique aux réactions désirées se sont révélés d'une efficacité modérée, de sorte que des méthodes rapides et rentables sont nécessaires pour générer des biocatalyseurs qui rendront la production chimique plus efficace. Dans l’ère de la bioinformatique et des outils de calcul pour soutenir l'ingénierie des enzymes, le développement rapide de nouvelles fonctions enzymatiques devient une réalité. Cette thèse commence par un examen des développements récents sur les outils de calcul pour l’ingénierie des enzymes. Ceci est suivi par un exemple de l’ingénierie des enzymes purement expérimental ainsi que de l’évolution des protéines. Nous avons exploré l’espace mutationnel d'une enzyme primitive, la dihydrofolate réductase R67 (DHFR R67), en utilisant l’ingénierie semi-rationnelle des protéines. La conception rationnelle d’une librarie de mutants, ou «Smart library design», impliquait l’association covalente de monomères de l’homotétramère DHFR R67 en dimères afin d’augmenter la diversité de la librairie d’enzymes mutées. Le criblage par activité enzymatique a révélé un fort biais pour le maintien de la séquence native dans un des protomères tout en tolérant une variation de séquence élevée pour le deuxième. Il est plausible que les protomères natifs procurent l’activité observée, de sorte que nos efforts pour modifier le site actif de la DHFR R67 peuvent n’avoir été que modérément fructueux. Les limites des méthodes expérimentales sont ensuite abordées par le développement d’outils qui facilitent la prédiction des points chauds mutationnels, c’est-à-dire les sites privilégiés à muter afin de moduler la fonction. Le développement de ces techniques est intensif en termes de calcul, car les protéines sont de grandes molécules complexes dans un environnement à base d’eau, l’un des solvants les plus difficiles à modéliser. Nous présentons l’identification rapide des points chauds mutationnels spécifiques au substrat en utilisant l'exemple d’une enzyme cytochrome P450 industriellement pertinente, la CYP102A1. En appliquant la technique de simulation de la dynamique moléculaire par la force de polarisation adaptative, ou «ABF», nous confirmons les points chauds mutationnels connus pour l’hydroxylation des acides gras tout en identifiant de nouveaux points chauds mutationnels. Nous prédisons également la conformation du substrat naturel, l’acide palmitique, dans le site actif et nous appliquons ces connaissances pour effectuer un criblage virtuel d'autres substrats de cette enzyme. Nous effectuons ensuite des simulations de dynamique moléculaire pour traiter l’impact potentiel de la dynamique des protéines sur la catalyse enzymatique, qui est le sujet de discussions animées entre les experts du domaine. Avec la disponibilité accrue de structures cristallines dans la banque de données de protéines (PDB), il devient clair qu’une seule structure de protéine n’est pas suffisante pour élucider la fonction enzymatique. Nous le démontrons en analysant quatre structures cristallines que nous avons obtenues d’une enzyme β-lactamase, parmi lesquelles un réarrangement important des résidus clés du site actif est observable. Nous avons réalisé de longues simulations de dynamique moléculaire pour générer un ensemble de structures suggérant que les structures cristallines ne reflètent pas nécessairement la conformation de plus basse énergie. Enfin, nous étudions la nécessité de compléter de manière informatisée un hémisphère où l’expérimental n’est actuellement pas possible, à savoir la prédiction de la migration des gaz dans les enzymes. À titre d'exemple, la réactivité des enzymes cytochrome P450 dépend de la disponibilité des molécules d’oxygène envers l’hème du site actif. Par le biais de simulations de la dynamique moléculaire de type Simulation Implicite du Ligand (ILS), nous dérivons le paysage de l’énergie libre de petites molécules neutres de gaz pour cartographier les canaux potentiels empruntés par les gaz dans les cytochromes P450 : CYP102A1 et CYP102A5. La comparaison pour les gaz CO, N2 et O2 suggère que ces enzymes évoluent vers l’exclusion du CO inhibiteur. De plus, nous prédisons que les canaux empruntés par les gaz sont distincts des canaux empruntés par le substrat connu et que ces canaux peuvent donc être modifiés indépendamment les uns des autres.
The chemical industry worldwide is at a turning point, seeking solutions to make classical organic synthesis more sustainable. One such solution is to shift from classical catalysis to biocatalysis. Although the advantages of enzymes include their stereo-, regio-, and chemoselectivity, their selectivity often reduces versatility. Past efforts to tailor enzymatic function towards desired reactions have met with moderate effectiveness, such that fast and cost-effective methods are in demand to generate biocatalysts that will render the production of fine and bulk chemical production more benign. In the wake of bioinformatics and computational tools to support enzyme engineering, the fast development of new enzyme functions is becoming a reality. This thesis begins with a review of recent developments on computational tools for enzyme engineering. This is followed by an example of purely experimental enzyme engineering and protein evolution. We explored the mutational space of a primitive enzyme, the R67 dihydrofolate reductase (DHFR), using semi-rational protein engineering. ‘Smart library design’ involved fusing monomers of the homotetrameric R67 DHFR into dimers, to increase the diversity in the resulting mutated enzyme libraries. Activity-based screening revealed a strong bias for maintenance of the native sequence in one protomer with tolerance for high sequence variation in the second. It is plausible that the native protomers procure the observed activity, such that our efforts to modify the enzyme active site may have been only moderately fruitful. The limitations of experimental methods are then addressed by developing tools that facilitate computational mutational hotspot prediction. Developing these techniques is computationally intensive, as proteins are large molecular objects and work in aqueous media, one of the most complex solvents to model. We present the rapid, substrate-specific identification of mutational hotspots using the example of the industrially relevant P450 cytochrome CYP102A1. Applying the adaptive biasing force (ABF) molecular dynamics simulation technique, we confirm the known mutational hotspots for fatty acid hydroxylation and identify a new one. We also predict a catalytic binding pose for the natural substrate, palmitic acid, and apply that knowledge to perform virtual screening for further substrates for this enzyme. We then perform molecular dynamics simulations to address the potential impact of protein dynamics on enzyme catalysis, which is the topic of heated discussions among experts in the field. With the availability of more crystal structures in the Protein Data Bank, it is becoming clear that a single protein structure is not sufficient to elucidate enzyme function. We demonstrate this by analyzing four crystal structures we obtained of a β-lactamase enzyme, among which a striking rearrangement of key active site residues was observed. We performed long molecular dynamics simulations to generate a structural ensemble that suggests that crystal structures do not necessarily reflect the conformation of lowest energy. Finally, we address the need to computationally complement an area where experimentation is not currently possible, namely the prediction of gas migration into enzymes. As an example, the reactivity of P450 cytochrome enzymes depends on the availability of molecular oxygen at the active-site heme. Using the Implicit Ligand Sampling (ILS) molecular dynamics simulation technique, we derive the free energy landscape of small neutral gas molecules to map potential gas channels in cytochrome P450 CYP102A1 and CYP102A5. Comparison of CO, N2 and O2 suggests that those enzymes evolved towards exclusion of the inhibiting CO. In addition, we predict that gas channels are distinct from known substrate channels and therefore can be engineered independently from one another.