Diss. (sammanfattning) Stockholm : Karol. inst. / Härtill 4 uppsatser.

Thesis (Ph. D.)--University of Texas at Austin, 2008. / Vita. Includes bibliographical references.

Microbial aromatic compound degradation often involves carbon-carbon bond hydrolysis of a meta-cleavage product (MCP). BphDLB400 (EC 3.7.1.8), the MCP hydrolase from the biphenyl degradation pathway of Burkholderia xenovorans LB400, hydrolyzes 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (HOPDA) to 2-hydroxypenta-2,4-dienoate (HPD) and benzoate. Although MCP hydrolases contain the catalytic triad (Ser112-His265-Asp237) and structural fold of the α/β-hydrolase superfamily, previous studies suggest they deviate from the classical hydrolytic mechanism in two respects: (1) enol-keto tautomerization precedes hydrolysis and (2) hydrolysis involves a gem-diol intermediate. Stopped-flow kinetic studies revealed rapid accumulation of a transient intermediate possessing a red-shifted absorption spectrum (λmax = 492 nm) versus HOPDA (λmax = 434 nm), consistent with an enzyme-bound, strained enolate (E:Sse). In studies with BphDLB400 variants, S112A trapped the E:Sse intermediate, implying that Ser112 is required for subsequent tautomerization and hydrolysis. His265 is required for E:Sse formation, as H265A variants instead generated a species assigned to a non-strained HOPDA enolate, which was not spectroscopically observed in the WT enzyme. The proposed importance of double bond strain in the reaction was supported by crystallographic observation of a non-planar, strained substrate in the S112A:HOPDA complex. Inhibition of BphDLB400 by 3-Cl HOPDA was investigated to understand a block in the degradation of polychlorinated biphenyls. BphDLB400 preferentially hydrolyzed 3-substituted HOPDAs in the order H > F > Cl > Me, indicating that steric bulk impairs catalysis. Kinetic analyses further indicated that large 3-substituents impede formation of the strained enolate by binding in an alternate conformation, as observed in the S112A:3-Cl HOPDA crystal structure. Finally, rate-determining hydrolysis of a benzoyl-enzyme was suggested from the observations that: (i) HOPDA and p-nitrophenyl benzoate were transformed with similar kcat values and (ii) yielded a common product ratio in the presence of methanol. Overall, the studies demonstrate the importance of an intermediate possessing significant double bond strain in an MCP hydrolase, establish the role of the catalytic His in forming this intermediate, indicate a mechanism of inhibition, and suggest the possibility that hydrolysis may proceed via an acyl-enzyme. / Medicine, Faculty of / Biochemistry and Molecular Biology, Department of / Graduate

No description available.

No description available.

No description available.

Nitrile-containing natural products are rare in Nature, and there have been very few studies on the mechanisms by which they are synthesized and utilized. The biosynthesis of 7-deazapurine containing natural products is a unique case whereby both formation of a nitrile and its conversion to an amide are documented. The overall theme of this work is to interrogate the biosynthesis of the nitrile intermediate in the pathway and its subsequent hydration to an amide. The biosynthesis 7-cyano-7-deazaguanine (preQ₀), the key intermediate in the biosynthesis of the hypermodified base queuosine and the toyocamycin natural product, is accomplished by preQ₀ synthetase through a series of unprecedented reactions whereby the carboxylate moiety of the substrate, 7-carboxy-7-deazaguanine (CDG), is successively activated by adenylation, reacted with ammonia, and dehydrated to produce the nitrile. This one-enzyme synthesis of a nitrile is unique as the only other known route to nitriles proceeds through at least two enzymes. Nitrile hydratases are metalloenzymes that selectively hydrate nitriles to the amide and are used industrially to produce acrylamide and nicotinamide. These enzymes use a trivalent iron or cobalt complex comprised of two backbone amidate ligands and three cysteine thiolate ligands of which two are modified to the sulfenato and sulfinato form. This work describes aspects of a particular nitrile hydratase, toyocamycin nitrile hydratase (TNH). Whereas most nitrile hydratases are heterodimeric, TNH is heterotrimeric, and yet what was discovered is that only the subunit containing the active site metal complex is required for activity. This single subunit analog of the protein was used for single turnover assays in ¹⁸O-labeled water to show with high resolution mass spectrometry that the source of the product amide oxygen is actually the enzyme itself and likely the sulfenato ligand oxygen acting as a nucleophile. The mechanism of the active site complex synthesis is described showing that this is self-catalytic in the presence of cobalt(II) and molecular oxygen.

by Yip Chi Wai. / Thesis (Ph.D.)--Chinese University of Hong Kong, 1998. / Includes bibliographical references (p. 94-113). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web. / Abstract in Chinese.

The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling.

The oligomerization of CPS from E. coli was investigated in order to examine the influence of this property on the catalytic activity. Mutations at the two interfacial sites of oligomerization were constructed in an attempt to elucidate the mechanism for assembly of the (αβ)4 tetramer through disruption of the molecular binding interactions between monomeric units. The results are consistent with a model for the structure of the (αβ)2 dimer that is formed through molecular contact between two pairs of allosteric domains. No significant dependence of the specific catalytic activity on the protein concentration could be detected. The molecular tunnel within CPS was inspected in order to characterize the role on kinetic properties. Gln-22, Ala-23, and Gly-575 from the large subunit of CPS were substituted by mutagenesis with bulkier amino acids in an attempt to obstruct and/or hinder the passage of the unstable intermediate through the carbamate tunnel. The kinetic data are consistent with a model for the catalytic mechanism of CPS that requires the diffusion of carbamate through the interior of the enzyme from the site of synthesis within the N-terminal domain of the large subunit to the site of phosphorylation within the C-terminal domain to yield a final product carbamoyl phosphate. In addition, a unique feature of the carbamate tunnel has been noted where five highly conserved glutamates are located on a particular interior face of the tunnel. It has been postulated that the negative charge stabilizes the acid-labile intermediate, and facilitates catalysis. Also, the proposed gate keeping residues, Arg-306 and Arg-848, have been mutated to alanines to test their roles. However, since the arginines directly interact with MgATP, the mutation appeared to interrupt the binding of the substrate. The ammonia tunnel has been engineered to contain a hole to further support the proposed role of the tunnel that it is utilized in guiding diffusion of ammonia from the site of glutamine hydrolysis to the subsequent active site in the large subunit. Triple mutant αP360A/αH361A/βR265A exhibited kinetic behaviors consistent with a model of an impaired channeling.