2013
DOI: 10.1074/jbc.m112.422535
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Characterization of the Enzyme CbiH60 Involved in Anaerobic Ring Contraction of the Cobalamin (Vitamin B12) Biosynthetic Pathway

Abstract: Background:The anaerobic biosynthesis of cobalamin involves contraction of a cobalt-containing porphinoid. Results: The contractase, CbiH 60 , contains a [4Fe-4S] center and catalyzes the conversion of cobalt factor III into cobaltprecorrin-4 in the presence of SAM and DTT. Conclusion: Ring contraction requires cobalt-precorrin-3 as a substrate. Significance: The high yield synthesis of cobalt-precorrin-4 will allow for further characterization of this pathway.

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Cited by 19 publications
(11 citation statements)
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“…This approach allowed significant quantities of soluble and relatively pure protein to be isolated. Furthermore, all these enzymes were stable in the standard buffers described (20 mM Tris·HCl, Hepes, pH 7-8, 100 mM NaCl), with the exception of CbiF and CbiH 60 (13), both of which required a high salt concentration (400 mM) to maintain solubility at 5-10 mg mL . The aggregation state of the various proteins was estimated by size exclusion chromatography (Table S2).…”
Section: Resultsmentioning
confidence: 99%
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“…This approach allowed significant quantities of soluble and relatively pure protein to be isolated. Furthermore, all these enzymes were stable in the standard buffers described (20 mM Tris·HCl, Hepes, pH 7-8, 100 mM NaCl), with the exception of CbiF and CbiH 60 (13), both of which required a high salt concentration (400 mM) to maintain solubility at 5-10 mg mL . The aggregation state of the various proteins was estimated by size exclusion chromatography (Table S2).…”
Section: Resultsmentioning
confidence: 99%
“…EPR has previously been used for the characterization of the early steps of the anaerobic B 12 pathway, including cobalt chelation into sirohydrochlorin by CbiX L (9), C-20 methylation by CbiL (12,19), and ring contraction reaction by CbiH 60 (13). For EPR, we shall now refer to each intermediate as cobalt(II)-precorrin-n, because all the pathway intermediates that we have analyzed are in the detectable paramagnetic cobalt(II) form.…”
Section: Resultsmentioning
confidence: 99%
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“…In addition, protein purification and secretion tools are available (33,34). From these tools, B. megaterium has recently been utilised to access challenging proteins (Fe-S, corrinoid enzymes), such as the reductive dehalogenase that were previously insoluble or inactive from using the routine "workhorse" E. coli (25,(35)(36)(37).…”
Section: Introductionmentioning
confidence: 99%