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, 101 (38), 13726-31

Amine-synthesizing Enzyme N-substituted Formamide Deformylase: Screening, Purification, Characterization, and Gene Cloning

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Amine-synthesizing Enzyme N-substituted Formamide Deformylase: Screening, Purification, Characterization, and Gene Cloning

Hiroshi Fukatsu et al. Proc Natl Acad Sci U S A.

Abstract

N-substituted formamide was produced through the hydration of an isonitrile by isonitrile hydratase in the isonitrile metabolism. The former compound was further degraded by a microorganism, strain F164, which was isolated from soil through an acclimatization culture. The N-substituted formamide-degrading microorganism was identified as Arthrobacter pascens. The microbial degradation was found to proceed through an enzymatic reaction, the N-substituted formamide being hydrolyzed to yield the corresponding amine and formate. The enzyme, designated as N-substituted formamide deformylase (NfdA), was purified and characterized. The native enzyme had a molecular mass of approximately 61 kDa and consisted of two identical subunits. It stoichiometrically catalyzed the hydrolysis of N-benzylformamide (an N-substituted formamide) to benzylamine and formate. Of all of the N-substituted formamides tested, N-benzylformamide was the most suitable substrate for the enzyme. However, no amides were accepted as substrates. The gene (nfdA) encoding this enzyme was also cloned. The deduced amino acid sequence of nfdA exhibited the highest overall sequence identity (28%) with those of regulatory proteins among known proteins. Only the N-terminal region (residues 58-72) of NfdA also showed significant sequence identity (27-73%) to that of each member of the amidohydrolase superfamily, although there was no similarity in the overall sequence except in the above limited region.

Figures

Fig. 1.
Fig. 1.
SDS/PAGE of the purified NfdA. Row A, the purified enzyme (4 μg); row B, marker proteins.
Fig. 2.
Fig. 2.
Sequence alignment of NfdA with distantly related proteins. Each region of the amino acid sequences of cytosine deaminase (CodA), dihydroorotase (PyrC), and urease α-subunit (UreC) contains a β-strand secondary structure in which the functional residues (histidine and aspartic acid) map to the C terminus of strands 1, 5, 6, and 8 in each enzyme (30). For NfdA, LAF3–1, HutI, and AtzA, the regions containing the predicted secondary structures that correspond to β1, 5, 6, or 8 of CodA, PyrC, and UreC are shown. LAF3 isoform 1 (LAF3–1) is from A. thaliana (GenBank accession no. AAP55749), AepA precursor (AepA) is from B. melitensis 16M (GenBank accession no. NP_541100.1), imidazolonepropionase (HutI) is from Caulobacter crescentus (SwissProt accession no. P58079), atrazine chlorohydrolase (AtzA) is from Pseudomonas sp. ADP (SwissProt accession no. P72156), cytosine deaminase (CodA) is from E. coli (SwissProt accession no. P25524), dihydroorotase (PyrC) is from E. coli (SwissProt accession no. P05020), and urease α-subunit (UreC) is from Klebsiella aerogenes (SwissProt accession no. P18314). The residues with amino acid numbers and vertical arrows are metal ligands established by x-ray crystallography of CodA (PDB ID code 1K6W), PyrC (PDB ID code 1J79), UreC (PDB ID code 2KAU), and the corresponding residues in the other proteins. The residues highlighted in reverse type are conserved in NfdA and all of the other proteins. Except for the residues highlighted in reverse type, identical amino acid residues in either of the regulatory proteins and the members of the amidohydrolase superfamily are boxed.
Fig. 3.
Fig. 3.
Schematic representation of NfdA and distantly related proteins. The five highly conserved residues are indicated, and the number for each histidine and aspartic acid residue indicates the deduced amino acid number in NfdA. Numbers on the right indicate the total numbers of amino acid residues in each molecule.

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