## 2 Results
### C. hircus UK114 Is a Deiminase  
The enzymatic activity of the purified recombinant protein was tested by a previouslypublished assay, which used l-leucine (l-Leu) as the substrate of the LAAO reaction ([6], Figure 3). This assay measures the decrease of the rate of (non-enzymatic) semicarbazone formation from the 2-imino acid produced by snake venom LAAO due to hydrolysis of the imine catalyzed by UK114 (Figure 3a). The addition of increasing amounts of UK114 in the assay decreased the production of semicarbazone in a dose-dependent manner, which is in agreement with the hydrolysis of the imino acid catalyzed by UK114 and its consequent subtraction from the reaction with semicarbazide (Figure 3b). For this reason, and for simplicity, we shall refer to this UK114 activity as a deiminase reaction instead of e  
Figure 2: Sequence alignment of UK114 with RidA proteins. Amino acid sequences of members of the RidA family from plants, insects, yeast, and bacteria were aligned using Clustal Omega at EMBI-EBI and shaded using the Expasy Box Shade server. From top the amino acid sequences and their UniprotKB accession number are: _Capra hircus_ UK114 (P80601), _Dermatoplagoides farinae_ Der f 34 (A0A1J1 DL12), _S. cerevisiae_ YER057c/_HMF1_ (P40037), and YIL051c/_MMF1_ (P40185), _A. thaliana_ RidA (Q94 JQ4), TdcF from _E. coli_ (P0 AGL2), and RidA from _Salmonella typhimurium_ (Q7 CP78). Similar residues are boxed in grey and identical residues are boxed in black. The stars indicate that the conserved residues of the active site and the diamond the residue that is not conserved with respect to the signature pattern of _E. coli_ TdcF [6]. Closed circles: residues required for oligomerization [19; 43]. Figure 3: Deiminase activity of UK114. **(a)** Scheme of the assay used to monitor UK114 deiminase activity. Activity assays of the deaminase activity of UK114 were carried out essentially as described by [6] and derived from [11]. The method exploits the formation of semicarbazone from the imino acid released from L-amino acid oxidase (LAAO) reaction in the presence of L-amino acids, which is four orders of magnitude faster than semicarbazone formation from the corresponding keto acid at pH 8.7 [11]. Thus, the activity of UK114 can be measured from the decrease of the velocity of semicarbazone formation. The possibility to substitute d-amino acid oxidase (DAAO) and d-amino acids for LAAO and L-amino acids was also tested in this work. The dashed arrows indicate the spontaneous hydrolysis of the imino acid, which competes with semicarbazone formation (**b**) Time-course of absorbance changes at 248 nm monitoring the formation of semicarbazone from semicarbazide (10 mM) and the imino acid produced by LAAO (11 \(\upmu\)g) and L-Leu (5 mM) in the absence (circles) or in the presence of UK114 (squares, 0.4 \(\upmu\)M; triangles, 3.9 \(\upmu\)M). The assays were carried out in 50 mM sodium pyrophosphate, pH 8.7, at 25 \({}^{\circ}\)C, in a final volume of 0.15 mL and included 1 \(\upmu\)g of catalase as in Ref. [6]. **(c)** Scatter graph of initial velocity values of semicarbazone formation from L-Leu (5 mM) and LAAO (11 \(\upmu\)g) in the presence of varying UK114 concentrations in 0.15 mL assays (open circles). The data from three independent experiments are shown along with those obtained in a similar experiment in which the imino acid was obtained from d-Leu and d-amino acid oxidase (closed circles). Error associated with measurements carried out under identical conditions was always less than 10%. An approximately linear relationship between the activity and the enzyme concentration was observed for UK114 concentrations up to 0.1 \(\upmu\)M. Greater than 80% decrease of the rate of semicarbazone formation was observed with approximately 4 \(\upmu\)M UK114 (Figure 3c). In principle, the imino acid could be produced also by DAAO from the corresponding D-amino acid provided its hydrolysis to the corresponding \(\upalpha\)-keto acid, and ammonia does not occur prior to the release from the enzyme active site.