Structural studies of biotechnologically relevant enzymes

Enzymes play an indispensable role in our everyday life. They are not only catalysing a vast number of reactions in our body, they are incorporated into many industrial processes. In the light of pollution and decline of fossil resources it is essential to develop new production routes to pharmaceutical and chemical target compounds. The exaggerated use of antibiotics and the resulting increase of multi‐resistant bacteria is another threat of our century. Solving this problem requires deep understanding of antibiotic resistance mechanisms to enable rational drug‐design. The main focus of the thesis lies on terpene synthases, which catalyse the cyclisation of linear isoprenoid precursors to complex macrocycles. These compounds are high commercial targets of the chemical and pharmaceutical industry. Terpenes constitute the largest group of natural compounds. Especially in the class of the diterpenes many compounds possess antibacterial, anti‐viral, insecticidal and antiinflammatory properties. White biotechnology opens up new routes for the production of terpenes in bacteria in a sustainable manner. In order to optimise terpene titers in bacteria, structural knowledge of the involved terpene synthases is crucial. Here, we report the crystal structures of the diterpene synthases CotB2 and IES in complex with various substrate‐analogues. Furthermore, the structure of the oleate hydratase OhyRe was elucidated. Products of this enzyme are used as surfactants and lubricants in industry. The X‐ray crystal structures of biotechnologically relevant enzymes obtained during this thesis contribute to illustrate their reaction mechanisms and binding modes. As another part of this thesis, the structure of AlbAS, a bacterial antibiotic‐binding protein, was solved. The protein structure sheds light on the binding mechanism of the antibacterial drug albicidin at a molecular level

Understanding the competing pathways leading to hydropyrene and isoelisabethatriene

Terpene synthases are responsible for the biosynthesis of terpenes, the largest family of natural products. Hydropyrene synthase generates hydropyrene and hydropyrenol as its main products along with two byproducts, isoelisabethatrienes A and B. Fascinatingly, a single active site mutation (M75L) diverts the product distribution towards isoelisabethatrienes A and B. In the current work, we study the competing pathways leading to these products using quantum chemical calculations in the gas phase. We show that there is a great thermodynamic preference for hydropyrene and hydropyrenol formation, and hence most likely in the synthesis of the isoelisabethatriene products kinetic control is at play

Towards a comprehensive understanding of the structural dynamics of a bacterial diterpene synthase during catalysis

Terpenes constitute the largest and structurally most diverse natural product family. Most terpenoids exhibit a stereochemically complex macrocyclic core, which is generated by C–C bond forming of aliphatic oligo-prenyl precursors. This reaction is catalysed by terpene synthases (TPSs), which are capable of chaperoning highly reactive carbocation intermediates through an enzyme-specific reaction. Due to the instability of carbocation intermediates, the proteins’ structural dynamics and enzyme:substrate interactions during TPS catalysis remain elusive. Here, we present the structure of the diterpene synthase CotB2, in complex with an in crystallo cyclised abrupt reaction product and a substrate-derived diphosphate. We captured additional snapshots of the reaction to gain an overview of CotB2’s catalytic mechanism. To enhance insights into catalysis, structural information is augmented with multiscale molecular dynamic simulations. Our data represent fundamental TPS structure dynamics during catalysis, which ultimately enable rational engineering towards tailored terpene macrocycles that are inaccessible by conventional chemical synthesis

Molecular insights into antibiotic resistance - how a binding protein traps albicidin

The worldwide emergence of antibiotic resistance poses a serious threat to human health. A molecular understanding of resistance strategies employed by bacteria is obligatory to generate less-susceptible antibiotics. Albicidin is a highly potent antibacterial compound synthesized by the plant-pathogenic bacterium Xanthomonas albilineans. The drug-binding protein AlbA confers albicidin resistance to Klebsiella oxytoca. Here we show that AlbA binds albicidin with low nanomolar affinity resulting in full inhibition of its antibacterial activity. We report on the crystal structure of the drug-binding domain of AlbA (AlbAS) in complex with albicidin. Both α-helical repeat domains of AlbAS are required to cooperatively clamp albicidin, which is unusual for drug-binding proteins of the MerR family. Structure-guided NMR binding studies employing synthetic albicidin derivatives give valuable information about ligand promiscuity of AlbAS. Our findings thus expand the general understanding of antibiotic resistance mechanisms and support current drug-design efforts directed at more effective albicidin analogs

Metal-triggered conformational reorientation of a self-peptide bound to a disease-associated HLA-B*27 subtype

Conformational changes of major histocompatibility complex (MHC) antigens have the potential to be recognized by T cells and may arise from polymorphic variation of the MHC molecule, the binding of modifying ligands, or both. Here, we investigated whether metal ions could affect allele-dependent structural variation of the two minimally distinct human leukocyte antigen (HLA)-B*27:05 and HLA-B*27:09 subtypes, which exhibit differential association with the rheumatic disease ankylosing spondylitis (AS). We employed NMR spectroscopy and X-ray crystallography coupled with ensemble refinement to study the AS-associated HLA-B*27:05 subtype and the AS-nonassociated HLA-B* 27:09 in complex with the self-peptide pVIPR (RRKWRRWHL). Both techniques revealed that pVIPR exhibits a higher degree of flexibility when complexed with HLA-B*27:05 than with HLA-B*27:09. Furthermore, we found that the binding of the metal ion Cu2+ or Ni2+, but not Mn2+, Zn2+, or Hg2+, affects the structure of a pVIPR-bound HLA-B*27 molecule in a subtype-dependent manner. In HLA-B*27:05, the metals triggered conformational reorientations of pVIPR, but no such structural changes were observed in the HLA-B*27:09 subtype, with or without bound metal ion. These observations provide the first demonstration that not only major histocompatibility complex class II, but also class I, molecules can undergo metal ion–induced conformational alterations. Our findings suggest that metals may have a role in triggering rheumatic diseases such as AS and also have implications for the molecular basis of metal-induced hypersensitivities and allergies

The Impression of a Nonexisting Catalytic Effect: The Role of CotB2 in Guiding the Complex Biosynthesis of Cyclooctat-9-en-7-ol

Terpene synthases generate terpenes employing diversified carbocation chemistry, including highly specific ring formations, proton and hydride transfers, and methyl as well as methylene migrations, followed by reaction quenching. In this enzyme family, the main catalytic challenge is not rate enhancement, but rather structural and reactive control of intrinsically unstable carbocations in order to guide the resulting product distribution. Here we employ multiscale modeling within classical and quantum dynamics frameworks to investigate the reaction mechanism in the diterpene synthase CotB2, commencing with the substrate geranyl geranyl diphosphate and terminating with the carbocation precursor to the final product cyclooctat-9-en-7-ol. The 11-step in-enzyme carbocation cascade is compared with the same reaction in the absence of the enzyme. Remarkably, the free energy profiles in gas phase and in CotB2 are surprisingly similar. This similarity contrasts the multitude of strong π–cation, dipole–cation, and ion-pair interactions between all intermediates in the reaction cascade and the enzyme, suggesting a remarkable balance of interactions in CotB2. We ascribe this balance to the similar magnitude of the interactions between the carbocations along the reaction coordinate and the enzyme environment. The effect of CotB2 mutations is studied using multiscale mechanistic docking, machine learning, and X-ray crystallography, pointing the way for future terpene synthase design