In their natural habitats, microorganisms have to frequently cope with a multitude of stressful biotic and abiotic conditions that can have adverse effects on their growth and their survival in a given ecosystem. One of the most important parameters of environmental stress due to its impact on almost all microorganisms is changing osmolarity/salinity. Among microorganisms, accumulation of compatible solutes is a widely used strategy to preserve cell integrity and growth under hyperosmotic conditions. The tetrahydropyrimidines ectoine and hydroxyectoine belong to these osmolytes and are produced by many procaryotes to minimize the adverse effects of high osmolarity on cellular physiology. Due to their beneficial impact on macromolecules, ectoine and hydroxyectoine are also reffered to in literature as chemical chaperones. These properties have spurred considerable biotechnological interest in ectoines.
Ectoine is enzymatically synthesized by the ectoine synthase (EctC) and the ectoine hydroxylase (EctD) catalyses the conversion of ectoine to hydroxyectoine. Despite the fact that both enzymes have already been studied somewhat, in-depth knowledge on their phylogenetic distribution, biochemistry and structure was still lacking prior to this dissertation since only a minor number of EctC and EctD proteins has been characterized and crystal structures of both proteins containing all ligands were still missing.
Since a deeper understanding of these key enzymes in ectoine biosynthesis is desirable, both with respect to basic science and industrial applications, the aim of the present dissertation was to assess the phylogenetic affiliation of ectoine biosynthetic genes and to study a selection of EctC and EctD enzymes with respect to their biochemical and kinetic properties. In addition, crystallographic approaches of EctC and EctD and site-directed mutagenesis experiments of EctD were conducted to provide a basis to unravel the position and binding motifs of the ligands within the catalytic cores of EctC and EctD.
To elucidate the phylogenetic distribution of ectoine biosynthesis, the amino acid sequences of both key enzymes were used as a search query and identified, after removal of redundant sequences, about 723 potential ectoine producers of which only 12 originated from Archaea. This analysis revealed that ectoine biosynthesis is widely distributed in prokaryotes, predominantly in members of the Bacteria, underlining the important role of ectoines in microbial stress responses.
On this basis, various ectoine synthases and ectoine hydroxylases deriving from different organisms have been biochemically and/or kinetically characterized that were widely distributed on the phylogenetic tree of ectoine biosynthesis. Each of the so far studied proteins possessed similar enzyme kinetics, however, comparison of their biochemical characteristics revealed only minor differences between EctD proteins but major variations between EctC enzymes. This suggests that the ectoine synthase, whose properties partially reflect the environmental circumstances of their hosts, might have developed in terms of evolution prior to the ectoine hydroxylase.
Identification of EctC and EctD proteins possessing increased stability allowed new crystallization trials. Multiple crystal structures have been solved in the course of this dissertation in collaboration with Dr. Sander Smits (University of Düsseldorf). In terms of EctD, structures in its apo-form, in complex with the iron co-factor and in complex with the iron catalyst, the co-substrate 2-oxoglutarate and the reaction product hydroxyectoine have been solved. These structures provided, in connection with comprehensive site-directed mutagenesis experiments, a detailed view into the catalytic core of EctD allowing a proposal for its catalyzed reaction mechanisms (Proposal by Dr. Wolfgang Buckel). In terms of EctC, a detailed expression and purification protocol for the ectoine synthase from the cold-adapted marine bacterium Sphingopyxis alaskensis has been described in collaboration with Dr. Sander Smits (University of Düsseldorf) that identifies EctC to form dimers in solution and deals with crystallization trials and preliminary X-ray diffraction data providing a promising basis for the solution of its crystal structure, which has, based on the presented data, been published posterior to the present dissertation.
Collectively, the present dissertation provides detailed information about the phylogenetic distribution and biochemistry of EctC and EctD as well as the solved crystal structure of the ectoine hydroxylase, the key enzymes in hydroxyectoine biosynthesis, and promising crystallization trials of EctC, the key enzyme in ectoine biosynthesis, and thus substantially contributes to the understanding of the role of ectoines in the global microbial osmostress adaptation.