The N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, the enzyme used for the industrial production of D-amino acids, was cloned, sequenced, and expressed in Escherichia coli. The protein, a dimer constituted by two identical subunits of 34,000 Da with five cysteines each, was susceptible to aggregation under oxidizing conditions and highly sensitive to hydrogen peroxide. To investigate the role of the cysteines in enzyme stability and activity, mutant proteins were constructed by site-directed mutagenesis in which the five residues were substituted by either Ala or Ser. Optically active D-amino acids have attained a wide variety of commercial applications as intermediates for the production of fine chemicals, including -lactam antibiotics, peptide hormones, and pesticides (1, 2). In particular, D-phenylglycine and D-p-hydroxyphenylglycine are among the most important chiral building blocks for the production of semisynthetic penicillins and cephalosporins such as ampicillin and amoxicillin.Several optically active D-amino acids are currently produced in a two-step reaction process starting from D,L-5 monosubstituted hydantoins that are inexpensively synthesized from the corresponding aldehydes (3). In the first step, the substrate is hydrolyzed by a D-specific hydantoinase to give a D-carbamyl derivative. Subsequently, the carbamyl derivative is converted to the corresponding D-amino acid either by chemical methods (4) or by a second enzymatic step catalyzed by an N-carbamyl-D-amino-acid amidohydrolase (hereinafter carbamylase) (5). Because chemical methods have high reaction temperatures, low yields, long reaction times, and generate large amounts of waste, the enzymatic hydrolysis of the N-carbamyl derivatives is highly preferred. Indeed, the use of the D-hydantoinase plus carbamylase two-enzyme system is considered one of the most successful industrial applications of enzyme technology.Several microorganisms expressing both enzymatic activities have been isolated, and the optimal reaction conditions and the biochemical properties of the two enzymes have been studied in some detail (6, 7). From what has been published so far, it appears that both the activity and stability of the carbamylase are negatively affected by oxidizing conditions, suggesting that one or more cysteine residues are present in the enzyme. Indeed, the sensitivity of the enzyme to oxidizing conditions is one of the most serious drawbacks of the enzymatic D-amino acid production process, and a strict anaerobic regime is required to allow the completion of the substrate to product conversion (8).To shed light on the role of the cysteines in the activity and stability of this important industrial enzyme, we decided to study in detail the N-carbamyl-D-amino-acid amidohydrolase from Agrobacterium radiobacter NRRL B11291, a strain that is used industrially for D-amino acid production. In this paper we describe the characterization of the recombinant A. radiobacter N-carbamyl-D-amino-acid amidohydrolase expresse...