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The 8-amino-7-oxopelargonate synthase from Bacillus sphaericus. Purification and preliminary characterization of the cloned enzyme overproduced in Escherichia coli.

ABSTRACT: The 8-amino-7-oxopelargonate synthase [6-carboxyhexanoyl-CoA:L-alanine carboxyhexanoyltransferase (decarboxylating); EC 2.3.1.47] from Bacillus sphaericus involved in biotin biosynthesis was purified from an Escherichia coli overproducing strain. The purification afforded an electrophoretically homogeneous enzyme with a specific activity of 0.67 unit/mg. The purified enzyme is a monomer of 41 kDa. N-Terminal sequencing of the first 14 amino acid residues showed complete agreement with the predicted sequence from the bioF gene. The pure enzyme showed the characteristic absorption band (425 nm) of pyridoxal 5'-phosphate-dependent enzymes. Furthermore, the holoenzyme was resolved during an affinity step yielding the inactive apoenzyme, which recovered activity and the 425 nm-absorption band on dialysis against pyridoxal 5'-phosphate. Km values for L-alanine and pimeloyl-CoA were respectively 3 mM and 1 microM.

SUBMITTER: Ploux O

PROVIDER: S-EPMC1131037 | biostudies-other | 1992 Apr

REPOSITORIES: biostudies-other

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A Canonical Biotin Synthesis Enzyme, 8-Amino-7-Oxononanoate Synthase (BioF), Utilizes Different Acyl Chain Donors in Bacillus subtilis and Escherichia coli.

Project description:BioF (8-amino-7-oxononanoate synthase) is a strictly conserved enzyme that catalyzes the first step in assembly of the fused heterocyclic rings of biotin. The BioF acyl chain donor has long been thought to be pimeloyl-CoA. Indeed, in vitro the Escherichia coli and Bacillus sphaericus enzymes have been shown to condense pimeloyl-CoA with l-alanine in a pyridoxal 5'-phosphate-dependent reaction with concomitant CoA release and decarboxylation of l-alanine. However, recent in vivo studies of E. coli and Bacillus subtilis suggested that the BioF proteins of the two bacteria could have different specificities for pimelate thioesters in that E. coli BioF may utilize either pimeloyl coenzyme A (CoA) or the pimelate thioester of the acyl carrier protein (ACP) of fatty acid synthesis. In contrast, B. subtilis BioF seemed likely to be specific for pimeloyl-CoA and unable to utilize pimeloyl-ACP. We now report genetic and in vitro data demonstrating that B. subtilis BioF specifically utilizes pimeloyl-CoA.IMPORTANCE Biotin is an essential vitamin required by mammals and birds because, unlike bacteria, plants, and some fungi, these organisms cannot make biotin. Currently, the biotin included in vitamin tablets and animal feeds is made by chemical synthesis. This is partly because the biosynthetic pathways in bacteria are incompletely understood. This paper defines an enzyme of the Bacillus subtilis pathway and shows that it differs from that of Escherichia coli in the ability to utilize specific precursors. These bacteria have been used in biotin production and these data may aid in making biotin produced by biotechnology commercially competitive with that produced by chemical synthesis.

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Cloning, purification, crystallization and preliminary crystallographic analysis of LsrR from Escherichia coli.

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Preliminary X-ray diffraction analysis of octaprenyl pyrophosphate synthase from Escherichia coli.

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Purification, crystallization and preliminary crystallographic analysis of a 6-pyruvoyltetrahydropterin synthase homologue from Esherichia coli.

Project description:6-Pyruvoyltetrahydropterin synthase from E. coli (ePTPS) has been crystallized using the hanging-drop vapour-diffusion method. Hexagonal- and rectangular-shaped crystals were obtained. Diffraction data were collected from the hexagonal and rectangular crystals to 3.0 and 2.3 A resolution, respectively. The hexagonal plate-shaped crystals belonged to space group P321, with unit-cell parameters a = b = 112.59, c = 68.82 A , and contained two molecules in the asymmetric unit. The rectangular crystals belonged to space group I222, with unit-cell parameters a = 112.76, b = 117.66, c = 153.57 A , and contained six molecules in the asymmetric unit. The structure of ePTPS in both crystal forms has been determined by molecular replacement.

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D-alanine: D-alanine ligase of Escherichia coli. Expression, purification and inhibitory studies on the cloned enzyme.

Project description:A 1.2 kb BamHI fragment from pDK30 [Robinson, Kenan, Sweeney & Donachie (1986) J. Bacteriol. 167, 809-817] was cloned in pDOC55 [O'Connor & Timmis (1987) J. Bacteriol. 169, 4457-4482] to give two constructs, pDOC89 and pDOC87, in which the Escherichia coli D-alanine:D-alanine ligase (EC 6.3.2.4) gene (ddl) was placed under the control of the lac and lambda PL promoters respectively. Both constructs, when used to transform E. coli M72, gave similar levels of expression of the ddl gene. The expressed enzyme was purified to homogeneity and the amino acid sequence of its N-terminal region was found to be consistent with that predicted from the gene sequence, except that the N-terminal methionine was not present in the mature protein. [1(S)-Aminoethyl][(2RS)2-carboxy-1-octyl]phosphinic acid (I), previously shown to bind tightly to Enterococcus faecalis and Salmonella typhimurium D-alanine:D-alanine ligases following phosphorylation Parsons, Patchett, Bull, Schoen, Taub, Davidson, Combs, Springer, Gadebusch, Weissberger, Valiant, Mellin & Busch (1988) J. Med. Chem. 31, 1772-1778; Duncan & Walsh (1988) Biochemistry 27, 3709-3714], was found to be a classical slow-binding inhibitor of the E. coli ligase.

| S-EPMC1130851 | biostudies-other

High-level expression of soluble rat hsc70 in Escherichia coli: purification and characterization of the cloned enzyme.

Project description:We have cloned the cDNA of rat hsc70 (clathrin-uncoating ATPase) into a T7 expression system and have expressed this enzyme in Escherichia coli. The recombinant clathrin-uncoating ATPase is in the cytosolic fraction of the bacterium and is soluble. It was purified to homogeneity by DEAE-cellulose and ATP-agarose column chromatography. From 1 litre of bacterial culture (0.3-0.4 g of proteins), 5-20 mg of pure recombinant clathrin-uncoating ATPase was routinely obtained. The cloned enzyme is capable of dissociating clathrin from bovine coated vesicle. Furthermore, it is not methylated on basic amino acid residues and is not blocked at the N-terminus, indicating that these modifications on hsc70 are not essential for uncoating of clathrin. Binding of [alpha-32P]ATP by purified recombinant hsc70 was analysed by Scatchard plot. The results indicate that there one high-affinity binding component with a Kd (dissociation constant) of 0.2-0.3 microM. The peptide-stimulated ATPase activities of recombinant hsc70 at 37 degrees C with respect to S-peptide peptides P3a and GT4 at a concentration of 1.2 mM are 142 +/- 6, 214 +/- 8 and 362 +/- 5 pmol/h per micrograms of hsc70 protein respectively. The EC50 values of hsc70 ATPase for S-peptide, peptides P3a and GT4 are 2, 0.67 and 0.17 mM respectively. On the other hand, the dissociation constants of S-peptide, peptides P3a and GT4 for recombinant hsc70 are 7.6, 13 and 100 microM respectively. Thus peptide GT4 is the only peptide examined for which the binding constant is comparable with the EC50 for stimulation ATPase activity, albeit it has the lowest affinity for hsc70.

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Expression, purification, crystallization and preliminary X-ray diffraction crystallographic study of PurH from Escherichia coli.

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Purification, crystallization and preliminary crystallographic analysis of the 16S rRNA methyltransferase RsmI from Escherichia coli.

Project description:RsmI and RsmH are AdoMet-dependent methyltransferases that are responsible for the 2'-O-methylation and N(4)-methylation of C1402 of Escherichia coli 16S rRNA, respectively. Modification of this site has been found to play a role in fine-tuning the shape and function of the P-site to increase the decoding fidelity. It is interesting to study the mechanism by which C1402 can be methylated by both RsmI and RsmH. The crystal structure of RsmH in complex with AdoMet and cytidine has recently been determined and provided some implications for N(4)-methylation of this site. Here, the purification and crystallization of RsmI as well as its preliminary crystallographic analysis are reported. Co-crystallization of RsmI with AdoMet was carried out by the sitting-drop vapour-diffusion method and X-ray diffraction data were collected to 2.60?Å resolution on beamline 1W2B at BSRF. The crystal contained three molecules per asymmetric unit and belonged to space group C2, with unit-cell parameters a = 121.9, b = 152.5, c = 54.2?Å, ? = 93.4°.

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High-level expression of rat PC12 tyrosine hydroxylase cDNA in Escherichia coli: purification and characterization of the cloned enzyme.

Project description:A rat cDNA containing the complete coding sequence for rat tyrosine hydroxylase (tyrosine 3-monooxygenase, EC 1.14.16.2) was isolated from a rat PC12 cDNA library and subcloned in a bacterial expression plasmid, and large amounts of functional enzyme were produced in Escherichia coli. The recombinant enzyme was purified approximately 20-fold to a final specific activity of 1.8 mumol/min per mg of protein, with a yield of 30%. As much as 1 mg of pure protein could be obtained from 1 g of wet bacterial cells. The purified hydroxylase was shown to be homogeneous by denaturing polyacrylamide electrophoresis and isoelectric focusing. Amino acid analysis of the N terminus (25 residues) revealed 100% identity with rat PC12 tyrosine hydroxylase, as deduced from its cDNA sequence. Several of the kinetic properties of the recombinant enzyme resembled those of the native PC12 hydroxylase. However, in contrast to the native enzyme, the purified recombinant hydroxylase was shown to be in an activated form. Phosphorylation with cAMP-dependent protein kinase resulted in stoichiometric incorporation of phosphate, but the kinetic profile of the recombinant enzyme was unaffected. Several clues to these differences are considered that may provide insight into the structural features important to the regulation of tyrosine hydroxylase.

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Purification, crystallization and preliminary crystallographic analysis of the 23S rRNA methyltransferase RlmM (Cm2498) from Escherichia coli.

Project description:RlmM is an AdoMet-dependent methyltransferase that is responsible for 2'-O-methylation of C2498 in the peptidyl-transferase loop of bacterial 23S rRNA. This modification occurs before assembly of the 50S ribosomal subunit, and lack of C2498 methylation can cause a slight reduction in bacterial fitness. Here, the purification and crystallization of RlmM from Escherichia coli as well as its preliminary crystallographic analysis are presented. Cocrystallization of RlmM with AdoMet was carried out and X-ray diffraction data were collected to a resolution of 2.30 Å on beamline BL17U at the SSRF. However, electron density for AdoMet cannot be observed by comprehensive crystallographic analysis, indicating that it is not bound by RlmM during the cocrystallization process. The structure was solved by molecular replacement and refinement is in progress. The crystal contained one molecule in the asymmetric unit and belonged to space group P2(1), with unit-cell parameters a = 56.07, b = 59.38, c = 54.35 Å, ? = 94.84°, which differs from the P3(1) or P3(1)21 space groups of previously reported RlmM structures (PDB entries 4auk, 4atn and 4b17).

| S-EPMC3668582 | biostudies-literature

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