Evaluation of substrate promiscuity of anL‐carbamoyl amino acid amidohydrolase fromGeobacillus stearothermophilusCECT43

Abstract

N-carbamoyl-amino-acid amidohydrolase (also known as N-carbamoylase) is the stereospecific enzyme responsible for the chirality of the D- or L-amino acid obtained in the "Hydantoinase Process." This process is based on the dynamic kinetic resolution of D,L-5-monosubstituted hydantoins. In this work, we have demonstrated the capability of a recombinant L-N-carbamoylase from the thermophilic bacterium Geobacillus stearothermophilus CECT43 (BsLcar) to hydrolyze N-acetyl and N-formyl-L-amino acids as well as the known N-carbamoyl-L-amino acids, thus proving its substrate promiscuity. BsLcar showed faster hydrolysis for N-formyl-L-amino acids than for N-carbamoyl and N-acetyl-L-derivatives, with a catalytic efficiency (k(cat)/K(m)) of 8.58 x 10(5), 1.83 x 10(4), and 1.78 x 10(3) (s(-1) M(-1)), respectively, for the three precursors of L-methionine. Optimum reaction conditions for BsLcar, using the three N-substituted-L-methionine substrates, were 65 degrees C and pH 7.5. In all three cases, the metal ions Co(2+), Mn(2+), and Ni(2+) greatly enhanced BsLcar activity, whereas metal-chelating agents inhibited it, showing that BsLcar is a metalloenzyme. The Co(2+)-dependent activity profile of the enzyme showed no detectable inhibition at high metal ion concentrations.

Role of Metal Ions on the Activity of Mycobacterium tuberculosis Pyrazinamidase

“…Previous studies have described metalloproteins that can use different metals as cofactors with equivalent efficiency. 18,[33][34][35][36] In previous reports, two independent groups have conducted very similar analyses on M. tuberculosis PZAse and the effects of divalent metals. The results produced by these two laboratories are largely divergent from those results reported here.…”

Mutational and Structural Analysis ofl-N-Carbamoylase Reveals New Insights into a Peptidase M20/M25/M40 Family Member

“…After purification, gel filtration chromatography was carried out using a Superdex 200 gel filtration column (GE Healthcare) in a BioLogic DuoFlow fast-performance liquid chromatography (FPLC) system (Bio-Rad) to eliminate any DNA or protein coeluting with the protein of interest. The purified enzymes were concentrated using Vivaspin concentrators (Sartorius), dialyzed against the optimum buffer, described previously (0.1 M sodium phosphate, pH 7.5) (48), and stored at 4°C until use. Protein concentrations were determined from the absorbance of extinction coefficient of tyrosine residues (19).…”

“…A standard enzymatic reaction was carried out with BsLcar and mutated enzymes, using a slightly modified method compared to that previously described (48). One hundred microliters of 5 M enzyme solution was preincubated with 1 mM CoCl 2 for 1 h. Four hundred microliters of 10 mM N-carbamoyl-L-methionine in 0.1 M sodium phosphate buffer (pH 7.5) was added and incubated at 50°C.…”

“…Due to their stereospecificity, L-carbamoylases are widely used in kinetic resolution for the production of optically pure amino acids (see reference 44 and references therein). Furthermore, their substrate promiscuity allows their use in different multienzymatic processes, increasing their economic and industrial relevance (48). L-Carbamoylases have been isolated and characterized from different sources, although most of the studies carried out have focused on biotechnological applications (44).…”

l-Amino Acid Production by a Immobilized Double-Racemase Hydantoinase Process: Improvement and Comparison with a Free Protein System

“…L-N-Carbamoyl amino acid amidohydrolase from Geobacillus stearothermophilus CECT43 (BsLcar) is a stereospecific enzyme with the capacity to hydrolyze N-acetyl Catalysts 2017, 7, 192 3 of 11 and N-formyl-L-amino acids as well as N-carbamoyl-L-amino acids, showing the best results for N-fomyl-L-amino acids. Optimal conditions are 65 • C and pH 7.5, and its activity increases with Co 2+ addition [12]. It is a stable enzyme that maintains 60% activity after incubation at 70 • C for one hour.…”