Partial Purification and Properties of d -Desthiobiotin Synthetase from Escherichia coli

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Abstract
D -Desthiobiotin synthetase, an enzyme that catalyzes the synthesis of d -desthiobiotin from dl -7,8-diaminopelargonic acid and HCO 3 − , was purified 100-fold from cells of a biotin mutant strain of Escherichia coli . Adenosine triphosphate and Mg 2+ were shown, especially in purified extracts, to be obligatory for enzyme activity, although concentrations higher than 5 m m caused severe inhibition of the reaction with unpurified cell-free extracts. Adenosine diphosphate and adenosine monophosphate were shown to inhibit the reaction, but fluoride (up to 50 m m ) had no detectable effect. The product of the enzyme reaction was identical to d -desthiobiotin on the basis of biological activity and chromatography. Furthermore, when H 14 CO 3 − was used as a substrate, the radioactive product was shown to be 14 C-desthiobiotin labeled exclusively in the ureido carbon.