Adenosylcobalamin-dependent methylmalonyl-CoA mutase from <i>Propionibacterium shermanii</i>. Active holoenzyme produced from <i>Escherichia coli</i>

McKie, Norman; Keep, Nicholas H.; Patchett, Mark L.; Leadlay, Peter F.

doi:10.1042/bj2690293

Cited by 48 publications

(55 citation statements)

References 44 publications

(41 reference statements)

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“…Cells containing pKSArg were grown aerobically at 37°C in M1 medium [17], supplemented with 10 mM glucose, 8 gM MnCI2 and 100 gg/ml ampicillin, to a culture OD600 of 2.0 and induced by adding IPTG to a final concentration of 0.25 raM. Cultures were grown for a further 4 h and cells harvested by centrifugation.…”

Section: Subcloning and Expression Of Arginasementioning

confidence: 99%

The cloning, expression and crystallisation of a thermostable arginase

et al. 1996

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The gene for the thermostable arginase from the thermophilic bacterium 'Bacillus caldovelox" has been cloned and sequenced. Expression of recombinant arginase at high levels has been achieved in E. coil using an inducible T7 RNA polymerasebased system. A facile purification procedure incorporating a heat-treatment step yielded 0.2 g of recombinant arginase per litre of induced culture. The kinetic properties of the purified recombinant protein are essentially identical to the native enzyme. The recombinant protein has been crystaHised and one crystal form is isomorphous to crystals of the native protein.

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Section: Subcloning and Expression Of Arginasementioning

confidence: 99%

The cloning, expression and crystallisation of a thermostable arginase

et al. 1996

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“…The C-terminal domain was partially insoluble after prolonged heat induction, but use of very brief heatshock treatment and subsequent incubation at a lower temperature than usually employed [26], allowed the protein to be obtained in a completely soluble form. After induction, it constituted about 20% of the total soluble protein (Fig.…”

Section: Mtsqellefths(v/h)vaail(g)xsxpdav; Is Consistent With the Expmentioning

confidence: 99%

An acyl‐carrier‐protein – thioesterase domain from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea

Caffrey

Green²,

Packman

et al. 1991

European Journal of Biochemistry

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The C‐terminal region of a multifunctional polypeptide from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea is predicted to contain an acyl carrier protein and a thioesterase or acyltransferase activity [Cortes, J., Haydock, S. F., Roberts, G. A., Bevitt, D. J. & Leadlay, P. F. (1990) Nature 348, 176–178]. Site‐directed mutagenesis by means of the polymerase chain reaction was used to construct an efficient pT7‐based expression plasmid for this domain. The recently developed technique of electrospray mass spectrometry was used to demonstrate that the purified protein had not been post‐translationally modified by attachment of a 4′‐phosphopantetheine group. However, treatment with the serine proteinase inhibitor phenylmethylsulphonyl fluoride led to highly selective labelling of the predicted active site of the thioesterase or acyltransferase.

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“…25, provided by P. Leadlay at the University of Cambridge) was subcloned into the pBS vector (Stratagene), which was then used as a template for creation of mutations by using the strategy supplied with the QuikChange kit (Stratagene). The following sense mutagenic primers were employed for PCR: H610A, GCCAGGACGGT-GCCGACCGCGGCCAGAAGGTC and H610N, GCCAGGACGGTAAC-GACCGCGGCCAGAAGGTC.…”

Section: Methodsmentioning

confidence: 99%

Importance of the Histidine Ligand to Coenzyme B12 in the Reaction Catalyzed by Methylmalonyl-CoA Mutase

Vlasie

Chowdhury

Banerjee

2002

Journal of Biological Chemistry

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Methylmalonyl-CoA mutase is an adenosylcobalamin (AdoCbl)-dependent enzyme that catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. The crystal structure of this protein revealed that binding of the cofactor is accompanied by a significant conformational change in which dimethylbenzimidazole, the lower axial ligand to the cobalt in solution, is replaced by His-610 donated by the active site. The contribution of the lower axial base to the ϳ10 12 -fold rate acceleration of the homolytic cleavage of the upper axial cobaltcarbon bond has been the subject of intense scrutiny in the model inorganic literature. In contrast, trans ligand effects in methylmalonyl-CoA mutase and indeed the significance of the ligand replacement are poorly understood. In this study, we have used site-directed mutagenesis to create the H610A and H610N variants of methylmalonyl-CoA mutase and report that both mutations exhibit both diminished activity (5,000-and 40,000-fold, respectively) and profoundly weakened affinity for the native cofactor, AdoCbl. In contrast, binding of the truncated cofactor analog, adenosylcobinamide, lacking the nucleotide tail, is less impaired. The catalytic failure of the His-610 mutants is in marked contrast to the phenotype of the adenosylcobinamide-GDP reconstituted wild type enzyme that exhibits only a 4-fold decrease in activity, although His-610 fails to coordinate when this cofactor analog is bound. Together, these studies suggest that His-610 may: (i) play a structural role in organizing a high affinity cofactor binding site possibly via electrostatic interactions with Asp-608 and Lys-604, as suggested by the crystal structure and (ii) play a role in catalyzing the displacement of dimethylbenzimidazole thereby facilitating the conformational change that must precede cofactor docking to the mutase active site.

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Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Active holoenzyme produced from Escherichia coli

Cited by 48 publications

References 44 publications

The cloning, expression and crystallisation of a thermostable arginase

The cloning, expression and crystallisation of a thermostable arginase

An acyl‐carrier‐protein – thioesterase domain from the 6‐deoxyerythronolide B synthase of Saccharopolyspora erythraea

Importance of the Histidine Ligand to Coenzyme B12 in the Reaction Catalyzed by Methylmalonyl-CoA Mutase

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