Crotonobetaine reductase from Escherichia coli consists of two proteins

Crotonobetaine reductase from Escherichia coli is composed of two proteins (component I (CI) and component II (CII)). CI has been purified to electrop horetic homogeneity from a cell-free extract of E. coli O44 K74. The purifi ed protein shows L(-)-carnitine dehydratase activity and its N-terminal ami no acid sequence is identical to the caiB gene product from E. coli O44 K74 . The relative molecular mass of CI has been determined to be 86100. It is composed of two identical subunits with a molecular mass of 42600. The isoe lectric point of CI was found to be 4.3. CII was purified from an overexpre ssion strain in one step by ion exchange chromatography on Fractogel EMD TM AE 650(S). The N-terminal amino acid sequence of CII shows absolute identit y with the N-terminal sequence of the caiA gene product, i.e, of the postul ated crotonobetaine reductase. The relative molecular mass of the protein i s 164400 and it is composed of four identical subunits of molecular mass 41 500. The isoelectric point of CLI is 5.6. CII contains non-covalently bound FAD in a molar ratio of 1:1. In the crotonobetaine reductase reaction one dimer of CI associates with one tetramer of CII. A still unknown low-molecu lar-mass effector described for the L(-)-carnitine dehydratase is also nece ssary for crotonobetaine reductase activity, Monoclonal antibodies were rai sed against the two components of crotonobetaine reductase. (C) 1999 Elsevi er Science B.V. All rights reserved.