Presentation Title

High-Throughput Mutagenesis Study of Phenol Hydroxylase to Engineer Alkane Hydroxylation Properties

Faculty Mentor

Matthew H. Sazinsky

Start Date

18-11-2017 10:00 AM

End Date

18-11-2017 11:00 AM

Location

BSC-Ursa Minor 50

Session

Poster 1

Type of Presentation

Poster

Subject Area

biological_agricultural_sciences

Abstract

Bacterial Multicomponent Monooxygenases (BMMs) are a class of enzymes characterized by their ability to hydroxylate a wide variety of hydrocarbon substrates. BMMs are naturally found in bacteria whose natural source of energy is derived from small organic compounds requiring initial hydroxylation for activation. BMMs are of particular interest to researchers because the substrate transformations performed can be useful in removing atmospheric greenhouse gases (i.e. methane) from the environment and in synthetic industrial applications. In this study, we attempt to develop a high-throughput mutagenesis screen that would allow us to engineer alkane-hydroxylating properties on Phenol Hydroxylase (PH). PH is a metalloenzyme coordinated by a carboxylate-bridged diiron center essential in carrying out oxidation on aromatic substrates (Phenols, Toluenes, etc.). However, this enzyme is unable to carry out oxidation on alkanes even though another BMM called soluble Methane Monooxygenase (sMMO) is able to do so. Various studies (sequence comparison, spectroscopic, and structural analysis) suggest that the primary coordination sphere is nearly identical in both BMMs, so it is of particular interest how both sMMO and PH carry out different chemical reactions. These studies suggest the differences in the chemistries is caused by differences in the secondary coordinating sphere of the diiron center. We hypothesize that it is an increased flexibility at the diiron center in sMMO that allows it to carry out alkane hydroxylation, and that mutating PH’s secondary sphere could activate alkane-hydroxylating properties in PH. In particular, a high-energy intermediate (Intermediate Q) is believed to allow sMMO to hydroxylate alkanes, but is inaccessible to PH. To carry out such a manipulation, we propose a high-throughput colorimetric screen for alkane hydroxylation of a randomly mutated library of the region surrounding PH’s active site. This approach will allow us to identify mutants PH that can perform alkane hydroxylation similar to sMMO's reaction.

Summary of research results to be presented

Previous approaches within this project involved creating single point mutations within the hydrolase component of the BMM followed by purification of the hydrolase (as well as the oxidoreductase and regulatory protein) to characterize the effects the mutation had on the enzyme. This process was very time-consuming and limited in scope. Therefore, we set out to develop a more powerful approach that would allow for a high-throughput colorimetric screening of E. coli cells expressing mutant forms of the BMM in vivo. This approach would eliminate the need to purify individual mutant proteins and could investigate many more mutations at a time. So far, we have been successful in creating the colorimetric screen. This involved developing an optimized technique for culturing bacterial cells expressing Phenol Hydroxylase’s full operon. We implemented a 96-deep well plate which could allow for the characterization of up to 96 mutants at once. We then developed our screen by testing various substrate candidates for out enzyme to hydroxylate. We identified two reactions that would result in colorimetric products. A catechol conversion assay that would tell us whether our enzyme maintains its native function and a 5-nitroguaiacol conversion assay that would identify alkane-hydroxylating mutants. The current stage of the project is focused on creating a mutant PH library by incorporating error-prone PCR to randomly mutate the region encoding for the secondary coordination sphere on PH. Upon creating our mutant library, we will transform mutant plasmids into E. coli and begin screening the resulting mutants for desired alkane hydroxylation.

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Nov 18th, 10:00 AM Nov 18th, 11:00 AM

High-Throughput Mutagenesis Study of Phenol Hydroxylase to Engineer Alkane Hydroxylation Properties

BSC-Ursa Minor 50

Bacterial Multicomponent Monooxygenases (BMMs) are a class of enzymes characterized by their ability to hydroxylate a wide variety of hydrocarbon substrates. BMMs are naturally found in bacteria whose natural source of energy is derived from small organic compounds requiring initial hydroxylation for activation. BMMs are of particular interest to researchers because the substrate transformations performed can be useful in removing atmospheric greenhouse gases (i.e. methane) from the environment and in synthetic industrial applications. In this study, we attempt to develop a high-throughput mutagenesis screen that would allow us to engineer alkane-hydroxylating properties on Phenol Hydroxylase (PH). PH is a metalloenzyme coordinated by a carboxylate-bridged diiron center essential in carrying out oxidation on aromatic substrates (Phenols, Toluenes, etc.). However, this enzyme is unable to carry out oxidation on alkanes even though another BMM called soluble Methane Monooxygenase (sMMO) is able to do so. Various studies (sequence comparison, spectroscopic, and structural analysis) suggest that the primary coordination sphere is nearly identical in both BMMs, so it is of particular interest how both sMMO and PH carry out different chemical reactions. These studies suggest the differences in the chemistries is caused by differences in the secondary coordinating sphere of the diiron center. We hypothesize that it is an increased flexibility at the diiron center in sMMO that allows it to carry out alkane hydroxylation, and that mutating PH’s secondary sphere could activate alkane-hydroxylating properties in PH. In particular, a high-energy intermediate (Intermediate Q) is believed to allow sMMO to hydroxylate alkanes, but is inaccessible to PH. To carry out such a manipulation, we propose a high-throughput colorimetric screen for alkane hydroxylation of a randomly mutated library of the region surrounding PH’s active site. This approach will allow us to identify mutants PH that can perform alkane hydroxylation similar to sMMO's reaction.