The glutaryl-7-aminocephalosporanic acid (GL-7-ACA) acylase isolated from Bacillus laterosporus J1 is capable of hydrolyzing GL-7-ACA and GL-7-ADCA to glutaric acid and the corresponding beta-lactam rings. Traditionally, J1 acylase was classified as class V of GL-7-ACA acylase (GCA). However, the amino acid sequence of J1 acylase has lower than 5% homology to acylases isolated from Pseudomonas strains. J1 acylase consists of a single peptide of molecular weight ∼78 kDa, in contrast with the heterodimeric nature of other cephalosporin acylases. Previous studies on this enzyme described only the specific activity, substrate preference, pH optimum and thermostability. Its tertiary structure and catalytic mechanism were not investigated in detail. It is interesting that the J1 acylase showed totally different structure from other classes of acylases but possessed the same hydrolytic activity towards cephalosporins. Homolog search revealed that J1 acylase showed 25% to 35% sequence identity to several alpha/beta-hydrolases including cocaine esterase (CocE) and alpha-amino acid ester hydrolases (AEHs). The putative catalytic triad residues conserved in J1 acylase were S125, D264 and H309, while the oxyanion-hole residues were Y57, Y126 and W173. The putative catalytic S125 was located within a highly conserved motif GXS&barbelow;YXG observed among S-15 peptidases. Secondary structure analysis had revealed alpha/beta-hydrolase fold at the N-terminal region. The catalytically important residues were located at positions where corresponding residues were found in alpha/beta-hydrolases. Tertiary structure was elaborated by homology modeling based on the X-ray structures of CocE and Acetobacter turbidans AEH (Pdb entries: 1JU3 and 1NX9, respectively). The models had demonstrated the three structural domains observed in CocE, with the putative catalytic triad residues positioned at the bottom of the active site cleft. Other catalytically important residues were identified according to the amino acid sequence alignments and residue superimpositions in tertiary structural model. Parallel site-directed mutagenesis experiments on these sites were performed to validate their functions. The mutants S125A, D264A, H309A, Y57A and Y57F were completely inactive to GL-7-ACA. Substituting Y126, V158, W173, W240 and L266 with an alanine resulted in decrease in catalytic efficiency. The two inflection points observed in the pH rate profile with pKa of 5.9 and 9.0 had indicated, respectively, that H309 and Y57 were involved in catalysis. Further kinetic and substrate spectrum studies had elucidated the substrate binding mechanism of J1 acylase. This study had demonstrated the previous classification of J1 acylase in the GCA classes is improper. I propose that this enzyme should be included in the alpha/beta-hydrolase superfamily evolved from the same origin of CocE and AEHs with catalytic activity towards cephalosporins. / by Yau Ming-hon. / "August 2005." / Adviser: Wang Jun. / Source: Dissertation Abstracts International, Volume: 67-07, Section: B, page: 3605. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2005. / Includes bibliographical references (p. 113-129). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. [Ann Arbor, MI] : ProQuest Information and Learning, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract in English and Chinese. / School code: 1307.

Thesis (M.S.)--Brandeis University, 2009. / Title from PDF title page (viewed on May 29, 2009). Includes bibliographical references.

No description available.

Thesis (Ph. D.)--University of Washington, 2003. / Vita. Includes bibliographical references (leaves 90-97).

This thesis describes an investigation into the folding behaviour of hen lysozyme by characterisation of the structure and the dynamics of different conformational states of the protein. The native state, a partially structured state generated by the addition of a cosolvent, trifluoroethanol (TFE), and a highly denatured state of the protein in presence of urea at low pH, have been studied at equilibrium by circular dichroism (CD) and nuclear magnetic resonance (NMR) spectroscopy. The principal methods utilised in-fchis thesis are the measurement and interpretation of amide hydrogen exchange and ¹⁵ N relaxation data. Amide hydrogen exchange rates from a highly denatured state of hen lysozyme in 8 M urea, at pH 2.0, are well approximated by recent estimates for intrinsic exchange rates from unstructured polypeptides, implying that residual .structures cannot be persistent in this state. In the native protein, by contrast, protection factors are measured to be of the order of 10⁴-10⁸ and can be rationalised by the involvement of amides in secondary structure and burial from the protein surface. Surface accessibility has also been found to be an important determinant of the dynamics of main and sidechain groups monitored by ¹⁵N relaxation. For this purpose ¹⁵N resonances of native lysozyme were assigned and order parameters derived. The dynamic behaviour was considered in the light of features of the crystal and NMR structures, suggesting that a lack of van der Waals contacts is a major determinant for mobility. A denatured state of hen lysozyme, formed by addition of TFE cosolvent, has been developed as a model for a partially ordered conformation of the protein. Amide hydrogen exchange measurements show that sites with the highest protection factors, up to 250, are located in stable native-like helices. Near complete assignment of ¹⁵N-edited NMR spectra allowed a detailed description of secondary structure in the TFE denatured state. Non-native α-helical structure is present as extensions to the native helices and in a region of the polypeptide which forms part of the β-sheet in the native state. These structures have been shown to be in accord with the helical propensities of the primary sequence. Preliminary structure calculations suggest that, despite the absence of extensive and persistent tertiary interactions, topological restraints exist in parts of the TFE denatured state, resulting in considerable propensities for native-like arrangements of the secondary structure. Mainchain dynamics determined by ¹⁵N relaxation, although increased in magnitude, appear to be related to that of the native state and are dominated by the position of disulphide bonds and by chain hydrophobicity. Thus the structural and the dynamic behaviour of the polypeptide chain in the TFE denatured state could be rationalised, at least in part, by consideration of features of the primary sequence.

Three different types (A, B, and C) of bile salt hydrolase from different Bifidobacterium strains revealed during the purification study showed the type-specific characteristics in their electrophoretic migration and elution profiles from anion exchange and hydrophobic interaction chromatographic columns. The subunit molecular mass estimated by SDS-PAGE was around 35 kDa and the native molecular mass in all types of BSH was estimated to be between 130 and 150 kDa by gel filtration chromatography, indicating that all BSH enzymes have tetrameric structure. From the isoelectric focusing, pI value of 4.45 was obtained with type B, but type A and C BSHs showed similar pI values of around 4.65. While the N-terminal amino acid sequences of types A and B were highly homologous (19/20), six out of twenty amino acid residues were different in the N-terminal sequences of types A and C. / As the type A bsh gene was cloned from a strain of B. longum and the nucleotide sequence became available from the GenBank, our study has focused on the cloning and characterization of the type B and C bsh genes from Bifidobacterium strains. / The type B bsh gene was cloned from B. bifidum ATCC11863 and the DNA flanking the bsh gene was sequenced. The 951 by-long bsh gene encoded a 316-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.48. For the first time in the genus Bifidobacterium, the transcriptional start point of the bsh gene was identified by primer extension analysis. Furthermore, Northern blot analysis revealed that B. bifidum bsh was transcribed as a monocistronic unit, contrary to that of B. longum bsh. Despite a high level of sequence similarity among the bsh genes, a BSH type-specific primer set based on the variable regions of bsh genes was designed in order to differentiate B. bifidum strains from the other species of Bifidobacterium commonly detected in the human gut. / The type C bsh gene was cloned from a bile salt tolerant strain of Bifidobacterium and the DNA flanking the bsh gene was further identified by a thermal asymmetric interlaced PCR (TAIL-PCR) technique. The 945 by-long bsh gene encoded a 314-amino-acid protein with a molecular mass of 35 kDa and a pI of 4.71. A predicted BSH promoter (Pbsh) sequence was experimentally verified and the transcriptional start point of the type C bsh gene was determined by primer extension analysis. An operonic structure including the type C bsh gene and two more ORFs, which were found within a complete set of a promoter and a transcription terminator, was identified in this study for the first time in the genus Bifidobacterium, and the polycistronic bsh transcript was revealed by RT-PCR and Northern blot analysis.

Thesis (Ph. D.)--University of Missouri-Columbia, 2006. / The entire dissertation/thesis text is included in the research.pdf file; the official abstract appears in the short.pdf file (which also appears in the research.pdf); a non-technical general description, or public abstract, appears in the public.pdf file. Vita. "December 2006" Includes bibliographical references.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

Photorhabdus é um gênero de enterobactérias simbiontes de Heterorhabditis, um gênero de nematoides entomopatogênicos. Dentre as enzimas secretadas por P. luminescens TTO1, destaca-se PrtA, uma metaloprotease que pertence a subfamília das serralisinas. PrtS, uma protease capaz de induzir uma forte resposta de melanização no inseto, foi identificada em P. temperata Az29 mas não em P. luminescens TTO1. Neste trabalho foram detectadas e caracterizadas bioquimicamente algumas proteases secretadas pelo isolado MN7 de P. luminescens luminescens. Foram detectadas duas atividades mais proeminentes: uma de 50 kDa, e outra de 38 kDa. Com o sequenciamento do genoma desta bactéria, pudemos confirmar a presença no genoma de genes codificando proteínas de alta identidade com as descritas PrtA e PrtS, de massas similares às detectadas nas nossas zimografias. As proteases são inibidas por inibidores específicos de metaloproteases. P. luminescens MN7 apresenta secreção, portanto, de uma protease já descrita algumas vezes, e de outra presente apenas em P. temperata Az29. / Photorhabdus is a genus of Enterobacteriaceae, symbionts of Heterorhabditis, a genus of entomopathogenic nematodes. Among the enzymes secreted by P. luminescens TTO1 stands out PrtA, a metalloprotease that belongs to the subfamily of serralysins. PrtS, a protease capable of inducing a strong melanotic response from the insect, was identified in P. temperata Az29 but not in P. luminescens TTO1. In this work were detected and characterized biochemically few isolated proteases secreted by P. luminescens luminescens MN7. Two most prominent activities were detected: one with 50 kDa and one with 38 kDa. With the sequencing of the genome of MN7, we could confirm the presence in the genome of genes encoding proteins with high identity to PrtA and PrtS already described, with similar masses to those detected in our zimographies. These proteases are inhibited by specific inhibitors of metalloproteases. P. luminescens MN7 secretes a protease already described a few times, and other present only in P. temperata Az29.

N-Acylethanolamines (NAEs) are endogenous lipid metabolites that occur in a variety of dry seeds, and their levels decline rapidly during the first few hours of imbibition (Chapman et al., 1999, Plant Physiol., 120:1157-1164). Biochemical studies supported the existence of an NAE amidohydrolase activity in seeds and seedlings, and efforts were directed toward identification of DNA sequences encoding this enzyme. Mammalian tissues metabolize NAEs via an amidase enzyme designated fatty acid amide hydrolase (FAAH). Based on the characteristic amidase signature sequence in mammalian FAAH, a candidate Arabidopsis cDNA was identified and isolated by reverse transcriptase-PCR. The Arabidopsis cDNA was expressed in E. coli and the recombinant protein indeed hydrolyzed a range of NAEs to free fatty acids and ethanolamine. Kinetic parameters for the recombinant protein were consistent with those properties of the rat FAAH, supporting identification of this Arabidopsis cDNA as a FAAH homologue. Two T-DNA insertional mutant lines with disruptions in the Arabidopsis NAE amidohydrolase gene (At5g64440) were identified. The homozygous mutant seedlings were more sensitive than the wild type to exogenously applied NAE 12:0. Transgenic seedlings overexpressing the NAE amidohydrolase enzyme showed noticeably greater tolerance to NAE 12:0 than wild type seedlings. These results together provide evidence in vitro and in vivo for the molecular identification of Arabidopsis NAE amidohydrolase. Moreover, the plants with altered NAE amidohydrolase expression may provide new tools for improved understanding of the role of NAEs in germination and seedling growth.