| dc.identifier.uri | http://hdl.handle.net/11401/77079 | |
| dc.description.sponsorship | This work is sponsored by the Stony Brook University Graduate School in compliance with the requirements for completion of degree. | en_US |
| dc.format | Monograph | |
| dc.format.medium | Electronic Resource | en_US |
| dc.language.iso | en_US | |
| dc.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dc.type | Dissertation | |
| dcterms.abstract | Mycobacterium tuberculosis (Mtb), as an intracellular pathogen, preferentially respires on lipids when surviving in vivo. Cholesterol metabolism is important for Mtb’s persistence and virulence. We demonstrate through experiment and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl-CoA dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four sub-pathways of cholesterol metabolism. Cholesterol side chain degradation is proposed to proceed through three β-oxidation cycles. ChsE1-ChsE2 (FadE28-FadE29) has been elucidated to function in the last cycle of β-oxidation through gene knockout studies and biochemical analysis. We identify and assign the substrate specificities of another two enzymes, ChsE4-ChsE5 (FadE26-FadE27) and ChsE3 (FadE34), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side-chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. Comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side-chain degradation by Mtb. Besides ACADs, we also characterized the enoyl-CoA hydratase function in the second step of the third cycle of β-oxidation. We solved the first structures of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, 3-oxo-4,17-pregnadiene-20-carboxyl-CoA, in the β-oxidation pathway for cholesterol side chain degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2N reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. The unique αβ heterodimer architecture utilized by ChsH1-ChsH2 to bind its distinctive substrate further highlights an opportunity for the development of new anti-mycobacterial drugs that target cholesterol metabolism pathway specific to Mtb. In addition, we investigated post-translational modification as a regulatory mechanism in the cholesterol metabolism pathway. We present a possible N-succinylase that is active with ChsE4-ChsE5 as a substrate, and demonstrate that introduction of a negative charge on lysine 238 reduces the catalytic activity of ChsE4-ChsE5. This modification in combination with other post-translational modifications in the pathway may suppress cholesterol metabolism under hypoxic conditions. We suggest that succinylation is a strategy for dynamically controlling this metabolic pathway to enable Mtb to quickly adapt to the changing environment via rapid sensing of the cellular redox status to flexibly alter reaction rates and directions. This demonstration provides us a new perspective to understand the metabolism of Mtb and adds an additional level to the complexity of the proteome. | |
| dcterms.abstract | Mycobacterium tuberculosis (Mtb), as an intracellular pathogen, preferentially respires on lipids when surviving in vivo. Cholesterol metabolism is important for Mtb’s persistence and virulence. We demonstrate through experiment and bioinformatic analysis the existence of an architecturally distinct subfamily of acyl-CoA dehydrogenase (ACAD) enzymes that are α2β2 heterotetramers with two active sites. These enzymes are encoded by two adjacent ACAD (fadE) genes that are regulated by cholesterol. Their structures and genomic locations suggest that the α2β2 heterotetrameric structural motif has evolved to enable catalysis of dehydrogenation of steroid- or polycyclic-CoA substrates and that they function in four sub-pathways of cholesterol metabolism. Cholesterol side chain degradation is proposed to proceed through three β-oxidation cycles. ChsE1-ChsE2 (FadE28-FadE29) has been elucidated to function in the last cycle of β-oxidation through gene knockout studies and biochemical analysis. We identify and assign the substrate specificities of another two enzymes, ChsE4-ChsE5 (FadE26-FadE27) and ChsE3 (FadE34), that carry out cholesterol side chain oxidation in Mtb. Steady-state assays demonstrate that ChsE4-ChsE5 preferentially catalyzes the oxidation of 3-oxo-cholest-4-en-26-oyl CoA in the first cycle of cholesterol side-chain β-oxidation that ultimately yields propionyl-CoA, whereas ChsE3 specifically catalyzes the oxidation of 3-oxo-chol-4-en-24-oyl CoA in the second cycle of β-oxidation that generates acetyl-CoA. However, ChsE4-ChsE5 can catalyze the oxidation of 3-oxo-chol-4-en-24-oyl CoA as well as 3-oxo-4-pregnene-20-carboxyl-CoA. The X-ray crystallographic structure of ChsE4-ChsE5 was determined to a resolution of 2.0 Å and represents the first high-resolution structure of a heterotetrameric acyl-CoA dehydrogenase (ACAD). Unlike typical homotetrameric ACADs that bind four flavin adenine dinucleotide (FAD) cofactors, ChsE4-ChsE5 binds one FAD at each dimer interface, resulting in only two substrate-binding sites rather than the classical four active sites. Comparison of the ChsE4-ChsE5 substrate-binding site to those of known mammalian ACADs reveals an enlarged binding cavity that accommodates steroid substrates and highlights novel prospects for designing inhibitors against the committed β-oxidation step in the first cycle of cholesterol side-chain degradation by Mtb. Besides ACADs, we also characterized the enoyl-CoA hydratase function in the second step of the third cycle of β-oxidation. We solved the first structures of a heterotetrameric MaoC-like enoyl-CoA hydratase, ChsH1-ChsH2, which is encoded by two adjacent genes from the igr operon. We demonstrate that ChsH1-ChsH2 catalyzes the hydration of a steroid enoyl-CoA, 3-oxo-4,17-pregnadiene-20-carboxyl-CoA, in the β-oxidation pathway for cholesterol side chain degradation. The ligand-bound and apoenzyme structures of ChsH1-ChsH2N reveal an unusual, modified hot-dog fold with a severely truncated central α-helix that creates an expanded binding site to accommodate the bulkier steroid ring system. The structures show quaternary structure shifts that accommodate the four rings of the steroid substrate and offer an explanation for why the unusual heterotetrameric assembly is utilized for hydration of this steroid. The unique αβ heterodimer architecture utilized by ChsH1-ChsH2 to bind its distinctive substrate further highlights an opportunity for the development of new anti-mycobacterial drugs that target cholesterol metabolism pathway specific to Mtb. In addition, we investigated post-translational modification as a regulatory mechanism in the cholesterol metabolism pathway. We present a possible N-succinylase that is active with ChsE4-ChsE5 as a substrate, and demonstrate that introduction of a negative charge on lysine 238 reduces the catalytic activity of ChsE4-ChsE5. This modification in combination with other post-translational modifications in the pathway may suppress cholesterol metabolism under hypoxic conditions. We suggest that succinylation is a strategy for dynamically controlling this metabolic pathway to enable Mtb to quickly adapt to the changing environment via rapid sensing of the cellular redox status to flexibly alter reaction rates and directions. This demonstration provides us a new perspective to understand the metabolism of Mtb and adds an additional level to the complexity of the proteome. | |
| dcterms.available | 2017-09-20T16:51:54Z | |
| dcterms.contributor | Raleigh, Daniel | en_US |
| dcterms.contributor | Sampson, Nicole S | en_US |
| dcterms.contributor | French, Jarrod | en_US |
| dcterms.contributor | Garcia-Diaz, Miguel. | en_US |
| dcterms.creator | Yang, Meng | |
| dcterms.dateAccepted | 2017-09-20T16:51:54Z | |
| dcterms.dateSubmitted | 2017-09-20T16:51:54Z | |
| dcterms.description | Department of Chemistry. | en_US |
| dcterms.extent | 259 pg. | en_US |
| dcterms.format | Monograph | |
| dcterms.format | Application/PDF | en_US |
| dcterms.identifier | http://hdl.handle.net/11401/77079 | |
| dcterms.issued | 2015-05-01 | |
| dcterms.language | en_US | |
| dcterms.provenance | Made available in DSpace on 2017-09-20T16:51:54Z (GMT). No. of bitstreams: 1
Yang_grad.sunysb_0771E_12441.pdf: 202885350 bytes, checksum: e8d898f77534a5aa9640fc7d8a20f9c8 (MD5)
Previous issue date: 2015 | en |
| dcterms.publisher | The Graduate School, Stony Brook University: Stony Brook, NY. | |
| dcterms.subject | Chemistry | |
| dcterms.subject | Acyl-CoA dehydrogenase, cholesterol metabolism, drug development, Enoyl-CoA hydratase, Tuberculosis | |
| dcterms.title | Identification and characterization of cholesterol metabolism related genes and gene products from Mycobacterium tuberculosis | |
| dcterms.type | Dissertation | |
