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Mycobacterium tuberculosis (Mtb) utilizes different metabolic pathways for its survival during infection. Enzymes of these pathways are often targets for antibiotic development. Genetic studies indicate the importance of the serine and biotin biosynthetic pathways for Mtb survival. In this study, enzymes from these pathways were characterized using X-ray crystallographic and biochemical studies. D-3-phosphoglycerate dehydrogenase (PGDH) catalyzes the first step of phosphorylated serine biosynthesis. In comparison to other forms of PGDH, the Mtb enzyme has an insertion near its C-terminus. This insertion results in two different conformations of the subunits in the tetramer, leading to two different environments for cofactor binding. This intervening domain might provide a second binding site for hydroxypyruvic acid phosphate (HPAP) that is responsible for substrate inhibition. Analysis of the HPAP-bound Mtb PGDH active site reveals the residues (Arg52, Arg131, and Arg233) involved in substrate interaction and provides insights into a possible enzyme mechanism. Mtb PGDH is feedback inhibited by the end product Lserine. Examination of the serine-bound PGDH structure elucidates the key players (Tyr461, Asp463, and Asn481) involved in this allosteric inhibition, as well as the resultant conformational changes at the regulatory domain interface. Preliminary biochemical studies of the first enzyme in Mtb biotin biosynthesis, 7- keto-8-aminopelargonic acid (KAPA) synthase show that it exists as a dimer in solution and has higher substrate affinity than the E. coli enzyme. The second enzyme, 7, 8-diaminopelargonic acid synthase (DAPAS) uses Sadenosyl methionine and KAPA as substrates in a bi-bi ping-pong mechanism. A comparison of the substrate analog sinefungin-bound Mtb DAPAS structure with a KAPA-bound DAPAS model provides a basis for the dual-substrate recognition. Tyr25 is a key player in the substrate specificity in DAPAS; this was confirmed by mutation studies. In certain Bacillus species, a Phe replaces this Tyr. The KAPA-bound B. subtilis DAPAS structure shows an alteration in the KAPA binding mode. Substrate and product bound structures of the third enzyme, dethiobiotin synthetase (DTBS) in Mtb reveal the important residues involved in its catalysis and provide framework for a possible enzyme mechanism. Comparison to the DTBS structures from E. coli and H. pylori reveals differences in local conformations.

Biologically active peptides are a class of molecules which can, in general, be grouped into families of structurally related moieties which have a wide somatic distribution and which subserve multiple physiological roles. Thus, the releasing factors of the hypothalamus or their structural analogues may carry out multiple endocrine, paracrine, or neurotransmitter functions outside the hypothalamus. This thesis describes the distribution and biosynthesis of Luteinizing Hormone-Releasing Hormone (LH-RH) and structurally related peptides in mammalian tissues and tumours. Two strategies were employed in this investigation: (a) Immunological screening of tissues for suitable sources of LH-RH and the partial characterization of the LH-RH immunoreactive peptides of selected tissues; (b) Complementary oligonucleotide screening of mRNA from certain tissues, cDNA and genomic libraries as well as genomic DNA restriction endonuclease fragments to establish the nature of the LH-RH precursor and related molecules. Acetic acid extracts of nineteen rat tissues were assayed in serial dilution for LH-RH immunoreactivity (LH-RH-IR). An antibody directed to the middle of the LH-RH sequence was used in these screening radioimmunoassays. LH-RH-IR was found in extrahypothalamic brain areas, in gastroenteropancreatic tissues, in the retina, the submandibular gland, the thyroid and the testes. In view of a putative role of endogeonous LH-RH-like immunoreactive peptides in intra-testicular regulation, the molecular nature of the LH-RH-like material detected in rat testes was investigated. Acetic acid extracts of adult rat testes were partially purified by LH-RH-immunoaffinity chromatography. On Sephadex G-100 this material separated into four major peaks of >100K, ~32K, ~5K and <4K daltons. The <4K peak of LH-RH-IR eluted differently on Sephadex G-25 and analytical reverse phase HPLC than did synthetic hypothalamic LH-RH decapeptide. Antibodies directed at the C-terminus of LH-RH gave higher estimates of LH-RH-IR for all the chromatographically separated testicular polypeptides than did N-terminally and middle directed antisera. The small testicular LH-RH-like peptides displaced radiolabelled LH-RH agonist from rat pituitary membranes more effectively than did the larger immunoreactive molecules. Monkey, pig and dog testes as well as dog Sertoli cell tumours were extracted with acetic acid and shown to contain LH-RH immunoreactive material similar to that of the rat. Human seminal plasma was also investigated as a source of LH-RH-IR. When pooled seminal plasma from azoospermic males was fractionated by gel filtration some fractions had significant amounts of the rat testicular type of LH-RH.

No description available.

No description available.

No description available.

No description available.

No description available.

No description available.