Hydrazidase, a Novel Amidase Signature Enzyme That Hydrolyzes Acylhydrazides

Abstract

The degradation mechanisms of natural and artificial hydrazides have been elucidated. Here we screened and isolated bacteria that utilize the acylhydrazide 4-hydroxybenzoic acid 1-phenylethylidene hydrazide (HBPH) from soils. Physiological and phylogenetic studies identified one bacterium as Microbacterium sp. strain HM58-2, from which we purified intracellular hydrazidase, cloned its gene, and prepared recombinant hydrazidase using an Escherichia coli expression system. The Microbacterium sp. HM58-2 hydrazidase is a 631-amino-acid monomer that was 31% identical to indoleacetamide hydrolase isolated from Bradyrhizobium japonicum. Phylogenetic studies indicated that the Microbacterium sp. HM58-2 hydrazidase constitutes a novel hydrazidase group among amidase signature proteins that are distributed within proteobacteria, actinobacteria, and firmicutes. The hydrazidase stoichiometrically hydrolyzed the acylhydrazide residue of HBPH to the corresponding acid and hydrazine derivative. Steady-state kinetics showed that the enzyme hydrolyzes structurally related 4-hydrozybenzamide to hydroxybenzoic acid at a lower rate than HBPH, indicating that the hydrazidase prefers hydrazide to amide. The hydrazidase contains the catalytic Ser-Ser-Lys motif that is conserved among members of the amidase signature family; it shares a catalytic mechanism with amidases, according to mutagenesis findings, and another hydrazidase-specific mechanism must exist that compensates for the absence of the catalytic Ser residue. The finding that an environmental bacterium produces hydrazidase implies the existence of a novel bacterial mechanism of hydrazide degradation that impacts its ecological role.

Functional Characterization of a Novel Amidase Involved in Biotransformation of Triclocarban and its Dehalogenated Congeners in Ochrobactrum sp. TCC-2

“…The stability of TccA meant its high tolerance to harsh conditions. Additionally, unlike the amidase HydA, which was induced by the substrate acylhydrazide, 44 TccA as well as other reported amidases, such as BbdA, LibA, and AmpA 24,33,42 were constitutively expressed. This suggests that tccA will be expressed under all conditions and not require TCC induction, making this strain useful for low-dose polluted sites.…”

Enzymatic production of key intermediate of gabapentin by recombinant amidase from Pantoea sp. with high ratio of substrate to biocatalyst

“…The amidase signature family enzymes possess a catalytic Ser-Ser-Lys triad [25], and these residues are conserved in Pa-Ami (Ser153, Ser177 and Lys80), Cc-Ami (Ser155, Ser179 and Lys82) and Dt-Ami 6 (Ser155, Ser179 and Lys82) (Fig. 2).…”

Draft Genome Sequence of Microbacterium sp. Strain HM58-2, Which Hydrolyzes Acylhydrazides

“…strain HM58-2 has been isolated from soil to utilize an acylhydrazide, 4-hydroxybenzoic acid 1-phenyletylidene hydrazide (HBPH). This strain produces intracellular hydrazidase, which is a member of the amidase family and is considered to be a key enzyme for hydrazide catabolism ( 1 ).…”

“…Since the expression of the hydrazidase gene is under catabolite repression, where it is induced by substrates, such as HBPH, as sole carbon source and repressed by the existence of glucose ( 1 ). Therefore, the genome sequence of strain HM58-2 will facilitate the understanding of this bacterium’s role in the catabolic mechanism of acylhydrazides.…”