Amine-synthesizing enzyme N-substituted formamide deformylase: Screening, purification, characterization, and gene cloning -- Fukatsu et al. 101 (38): 13726 Data Supplement - HTML Page - index.htslp -- Proceedings of the National Academy of Sciences

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Fukatsu et al. 10.1073/pnas.0405082101.

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Supporting Materials and Methods
Supporting Figure 4
Supporting Table 2
Supporting Figure 5
Supporting Figure 6

Supporting Figure 4

Fig. 4. Effects of pH and temperature on the activity of N-substituted formamide deformylase. (A) Reactions were carried out for 10 min at 25°C in the following buffers: 0.1 M citrate-Na₂HPO₄ (▲), 0.1 M potassium phosphate (○), 0.1 M Tris/HCl buffer (●), and 0.1 M NH₄OH-NH₄Cl (∆). (B) Reactions were carried out for 10 min at various temperatures. Relative activity is expressed as the percentage of the maximum activity attained under the experimental conditions used.

Table 2. Substrate specificity of N-substituted formamide deformylase (NfdA)

Substrate	Relative activity, %
N-benzylformamide	100.00
N-butylformamide	3.40
Allylformamide	0.22
N-(2-cyclohex-1-enylethyl)formamide	0.13
N-(α-methylbenzyl)formamide	0.16

All experiments were carried out by using the assay conditions described in Materials and Methods. The activity is given relative to the deformylation of N-benzylformamide.

Supporting Figure 5

Fig. 5. Alignment of the amino acid sequences of N-substituted formamide deformylase (NfdA) and regulatory proteins. Residues that are conserved between NfdA and either of the regulatory proteins (LAF3-1 and AepA) are highlighted in reverse type. Arrows indicate the residues corresponding to highly conserved His and Asp residues in the amidohydrolase superfamily.

Supporting Figure 6

Fig. 6. Secondary structure prediction of NfdA and regulatory proteins. The structural predictions were performed with the program PSI-PRED and PROFSEC. The predictions for secondary structure (PSIPRED; RROF_sec) (H, a -helix; E, b -strand), the confidence of PSI-PRED prediction (Confidence), and the reliability of PROFSEC prediction (Rel_sec) for four regions of NfdA, LAF3-1, and AepA, are shown. The confidence or reliability of these predictions varies between 0 (low) and 9 (high). For a comparison, the common b secondary structures (b 1, b 5, b 6, and b 8) of CodA, PyrC, and UreC are shown at the bottom. The residues shown in bold are highly conserved amino acids in the amidohydrolase superfamily and the corresponding residues in NfdA, LAF3-1, and AepA. Numbers in parentheses are the length of the intervening sequence.