Mechanistic Investigations of a Novel Flavin-dependent Enzyme Involved in Styrene Biosynthesis

Abstract

The development of biorenewable resources to substitute for oil-based feedstocks in polymer manufacture is essential to address sustainability. Ethylene, propylene, and styrene together represent ~90% of the feedstocks used by the polymer industry. Some industries have attempted to utilize bio-based ethylene and propylene. However, there are very few known biosynthetic pathways to produce styrene and, generally, they are not well understood. The decarboxylation of cinnamic acid by ferulic acid decarboxylase (FDC) is one of the few biological pathways known that form styrene. The focus of this dissertation is to explore the recently proposed FDC decarboxylation mechanisms and cofactor specificity of the enzyme. The initially proposed mechanisms for FDC decarboxylation of phenylacrylic acid postulated either Michael addition or 1,3-dipolar cycloaddition. 1H NMR analysis, isotope effects and linear free-energy analysis were employed to further investigate the proposed mechanisms and determine the rate-determining step in the reaction. 1H NMR experiments demonstrated that FDC decarboxylation was stereospecific and the source of proton that replaced the carboxylic group of phenylacrylic acid was the solvent. Proton inventory experiments suggested that a single proton was involved in the transition state. The negative Hammett reaction constant provided evidence that the 1,3-cycloelimination step in the 1,3-dipolar cycloaddition mechanism is likely to be rate-determining in the FDC decarboxylation. This was further supported by secondary kinetic isotope effect experiments. xv To investigate the biosynthesis of the prFMN cofactor by prenyl flavin synthase (PFS), a scPFSFDC coupled assay was developed and optimized to monitor scPFS prenylation through the activation of FDC. With scPFS-FDC coupling assay, we found that scPFS selectively uses dimethylallyl pyrophosphate as the substrate for FMN prenylation, in contrast to the bacterial enzyme for which dimethylallyl monophosphate was reported to be the substrate. By implementing this coupled assay, steady-state kinetic parameters of scPFS prenylation were obtained. Commercially available substrate analogs, (E)-4-Hydroxy-3-methyl-but-2-enyl pyrophosphate (HMBPP) and geranyl pyrophosphate (GPP), were used to investigate the substrate specificity of scPFS prenylation. The apparent first-order rate constants of prenylation were 4.45 ± 0.87 h-1, and 3.80 ± 0.34 h-1 when HMBPP and GPP were respectively used as substrate under single turnover kinetics. Under steady state conditions, FDC was shown to utilize the cofactor synthesized from HMBPP to decarboxylate cinnamic acid at Vmax = 5.50 ± 0.37 min-1. However, in the case of GPP counterpart, no decarboxylation activity was observed. The results presented in this thesis provide better understanding of styrene biosynthesis by FDC. In order to be implemented in bioindustry, this system would have to be further optimized, especially in terms of enzymatic efficiency. One proposed strategy is the use of cofactor analogs. Preliminary investigations here have enabled to establish the procedure that would ultimately lead to the discovery of cofactor analogs with enhanced decarboxylation activity.