Date of Award

2015

Degree Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Chemistry

Abstract

GabR is a member of the MocR/GabR subfamily of the GntR family of bacterial transcription regulators. It regulates the metabolism of γ-aminobutyric acid (GABA), an important nitrogen and carbon source in many bacteria. The crystal structures reported here show that this protein has evolved from the fusion of a type I aminotransferase and a winged helix-turn-helix (wHTH) DNA binding protein to form a chimeric protein that adopts a dimeric head-to-tail configuration. The pyridoxal 5′-phosphate (PLP)-binding regulatory domain of GabR is therefore an example of a coenzyme playing a role in transcription regulation rather than in enzymatic catalysis. Our structural and biochemical studies lay the mechanistic foundation for understanding the regulatory functions of the MocR/GabR subfamily of transcription regulators.

GabR is also an intriguing case of molecular evolution, displaying the evolutionary lineage between a PLP-dependent aminotransferase and a regulation domain of a transcription regulator. Here, PLP's native function is not a catalytic co-enzyme but an effector of transcription regulation. The chemical species of GabR-PLP-GABA, which is responsible for GabR-mediated transcription activation, has been revealed as a stable external aldimine formed between PLP and GABA by a crystal structure with further support from results in mechanistic crystallography, NMR spectroscopy and biological assays using both GABA and a GABA analog (S)-4-Amino-5-Fluoropentanoic Acid (AFPA) as a molecular probe. Our studies provide critical structural and chemical insights for a currently understudied subfamily of bacterial transcription regulators, MocR/GabR-type regulators in the GntR family.

In paralle, I also worked on several other projects. Herein, I want to dedicate 2 full chapaters to demonstrate the progress on the Sucrose Synthase projct. We obtained biochemical and structural evidence of sucrose metabolism in non-photosynthetic bacteria. Until now, only sucrose synthases from photosynthetic organisms have been characterized. Here, we provide the crystal structure of the sucrose synthase from the chemolithoautotroph Nitrosomonas europaea. This supported that the enzyme functions with an open/close induced fit mechanism. It prefers as substrate adenine-based nucleotides rather than uridine-based like the eukaryotic counterparts, implying there is a strong connection between sucrose and glycogen metabolism in these bacteria. Mutagenesis data showed that the catalytic mechanism must be conserved not only in sucrose synthases, but also in all other retaining GT-B glycosyltransferases.

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