Assessing the stability of aquatic extracellular enzymes

Abstract

Aquatic extracellular enzymes are important drivers of organic matter cycling in surface waters. Heterotrophic bacteria secret extracellular enzymes to break down macromolecular carbon into bioavailable substrates. Once secreted, these enzymes are exposed to different environmental conditions and undergo transformation processes which can compromise enzyme stability and consequently alter their affect on biogeochemical cycles. The activity of an enzyme critically depends on the molecular integrity of the active site residues. Simultaneous assessment of transformations in the chemical structure and enzyme inactivation can lead to a better understanding of the molecular processes that lead to enzyme inactivation. Photochemical damage is one environmental process that can inactivate extracellular enzymes in surface waters. A compound degrades via direct photochemical processes when it absorbs light directly and via indirect processes when it reacts with photochemically produced reactive intermediates. The indole moiety of tryptophan residues absorbs light in the UVB range and represents the major chromophore in amino acid-based molecules. Tryptophan photoionization leads to tryptophan degradation and initiates subsequent intramolecular reaction such as tyrosine oxidation and disulphide reduction. Indirect photochemical degradation of proteins with singlet oxygen (1O2), an important oxidant in surface waters, potentially leads to transformation of the five photolabile amino acids tryptophan, tyrosine, methionine, histidine and cysteine. Cyanopeptides are another, though less explored, environmental factor that potentially influences extracellular enzyme stability. Fast cell proliferation in cyanobacterial blooms induces successive growth of heterotrophic bacteria leading to the co-occurrence of cyanopeptides and extracellular enzymes. Cyanopeptides reach high concentrations in cyanobacterial blooms posing risks to human and ecological health. Cyanopeptides have a high structural diversity and can be divided in multiple classes including microcystins, cyanopeptolins, anabaenopeptins, aerucyclamides, aeruginosins and microginins. So far, research has mainly focused on the class of microcystins although in blooms cyanopeptides belonging to other classes have been shown to be co-produced in similar concentrations. Enzyme inhibition is a commonly observed effect of cyanopeptides which has been studied for some human toxicologically relevant proteases and a liver protein phosphatase. Effects on biogeochemically relevant extracellular enzymes have not been reported to date. The overall goal of this work was to assess the stability of aquatic extracellular enzymes towards the two environmental factors light and secondary metabolites produced by cyanobacteria. This work involved the development of analytical approaches to study site-specific molecular changes in the three model enzymes that catalyze reactions in different biogeochemical cycles: E. coli alkaline phosphatase, A. proteolytica leucine aminopeptidase and B. stearothermophilus alpha-glucosidase. In the first part of this thesis, I characterized the direct photochemical degradation on a molecular level by observing site-specific changes in model enzymes and on a functional level by activity measurements. Exposure to UVB light led to fast direct photochemical enzyme degradation by tryptophan photoionization which triggered subsequent intramolecular reactions. A spatio-temporal analysis revealed that the reactivity of a residue is substantially influenced by the local environment. The occurrence of subsequent reactions was dependent on the presence of and the inter-residue distance between the reaction partners, which is defined by the macromolecular structure. I found evidence for subsequent reduction of tryptophan radical cation by phenolic tyrosine, disulfide reduction and methionine oxidation mediated by triplet state tryptophan. In the second part of this work, I assessed indirect photochemical inactivation focussing on the oxidant 1O2. I inspected site-specific amino acid transformation kinetics and product formation in phosphatase and aminopeptidase by a detailed analysis of amino acid transformation products and pathways. The time-resolved sampling facilitated a comprehensive kinetic analysis and revealed a complex network of 1O2-induced reaction pathways that explained the observed enzyme degradation. Transformation products were identified using a semi-automated suspect screening that filtered for masses showing formation kinetics and validated by isotope pattern and MS2 fragmentation information. The results of the transformation product pathway analysis indicated that the 1O2-reactivity of proteinogenic amino acids is influenced by the local environment of the residue but that solvent exposure, often correlated with accessibility to the oxidant, is not necessarily an accurate predictor for the site-specific reactivity. I observed influences of the local environment on site-specific reactivity by interactions with metal co-factors for histidine or intramolecular hydrogen bonding for tyrosine. The developed approach for a holistic assessment of site-specific transformation and pathway analysis can be applied to study 1O2-induced degradation of other proteins such as cellular proteins that are exposed to high intracellular 1O2 concentrations. Furthermore, the presented approach can also be used to study other processes that produce distinct amino acid transformation products such as post-translational modifications. In the last part of this thesis, I present results on inhibition of aquatic extracellular enzymes by cyanopeptide extracts from different cyanobacteria strains and purified cyanopeptides. Bacterial leucine aminopeptidase showed considerable inhibition by exposure to the apolar fractions of M. aeruginosa, M. aeruginosa mutant and P. rubescens extracts as well as to purified cyanopeptolin A, aerucyclamide A in comparable concentrations as present in the tested extracts. Inhibition was also observed for bacterial phosphatase and a microcystin-free extract showed strongest effects. These findings show that cyanobacterial metabolites can have inhibitory effects on biogeochemically relevant enzymes. In summary, the work presented herein improved our understanding of extracellular enzyme stability towards different environmental factors and advanced analytical methods to study site-specific changes as well as enzyme activity.