(228c) Structural Insights into Catalytic Function of Non-P450 Hydroxylase (HpaB) from Escherichia coli

Authors: 
Shen, X., University of Georgia
Lin, Y., BiotecEra Inc.
Zhou, D., University of Georgia
Wang, J., University of Georgia
Yuan, Q., Beijing University of Chemical Technology
Rose, J., University of Georgia
Yan, Y., University of Georgia
Hydroxylation plays a pivotal role in the synthesis of various natural products. Regio-specific hydroxylation is usually difficult to be achieved via chemical synthesis approaches due to its low selectivity and requirement for excitation, protection and de-protection of other groups. In biological systems, a number of cytochrome P450 hydroxylases can catalyze hydroxylation of inert carbon. However, it is hardly used in metabolic engineering owing to its low stability, lower activity and the requirement for a reductase partner. 4-hydroxyphenylacetate 3-hydroxylase (HpaB) is a flavin adenine dinucleotide (FAD) dependent hydroxylase existing in Escherichia. coli. It has broad substrate spectrum and can efficiently catalyze ortho-hydroxylation towards a series of phenylpropanoids to produce the hydroxyl derivatives, while the latter are widely used in clinical research and pharmacological industry. In this work, a structure-based study is conducted to expand the catalytic function of HpaB. Firstly, we elucidated the crystal structure of HpaB by X-ray crystallography. Based on the structure, we predicted that a loop covering the entrance of the catalytic site may play a significant role in substrate selection and catalytic activity. In order to extend the substrate spectrum and improve the catalytic efficiency to large phenylpropanoid molecules, seven variants have been generated to change the rigidity of the loop structure. Four different substrates with different molecular sizes, including p-coumaric acid, 7-hydroxycoumarin, resveratrol and naringenin, were used to test the activity of those variants. We found one mutant enzyme with the most flexible spatial conformation in the loop structure has shown the highest catalytic activity towards all four substrates. In vivo bioconversion and in vitro enzyme assay of the mutant indicated that the conversion rate was increased by 80% and the Km value was decreased dramatically towards the largest substrate naringenin compared with wild type HpaB. Especially, this mutant exhibited the highest ortho-hydroxylation activity towards naringenin reported so far. These results suggest that the flexible nature of the loop is an important determinant for the enzyme to accommodate larger molecules. This research increases the substrate accommodations and improves the activity of hydroxylase HpaB via structure-based rational design, which implied potential application in metabolic engineering of large phenylpropanoids and provided new thoughts in protein engineering of spatial conformation of the enzyme.