Highly stable meso-diaminopimelate dehydrogenase from an Ureibacillus thermosphaericus strain A1 isolated from a Japanese compost: purification, characterization and sequencing

Abstract

We screened various thermophiles for meso-diaminopimelate dehydrogenase (meso-DAPDH, EC 1.4.1.16), which catalyzes the NAD(P)-dependent oxidative deamination of meso-diaminopimelate, and found the enzyme in a thermophilic bacterium isolated from compost in Japan. The bacterium grew well aerobically at around 55°C and was identified as Ureibacillus thermosphaericus strain A1. We purified the enzyme about 47-fold to homogeneity from crude cell extract using five successive purification steps. The molecular mass of the purified protein was about 80 kDa, and the molecule consists of a homodimer with the subunit molecular mass of about 40 kDa. The optimum pH and temperature for the catalytic activity of the enzyme are about 10.5 and 65°C, respectively. The enzyme is highly selective for meso-diaminopimelate as the electron donor, and NADP but not NAD can serve as the electron acceptor. The Km values for meso-diaminopimelate and NADP at 50°C and pH 10.5 are 1.6 mM and 0.13 mM, respectively. The nucleotide sequence of this meso-DAPDH gene encodes a 326-amino acid peptide. When the gene was cloned and overexpressed in Escherichia coli Rosetta (DE3), the specific activity in the crude extract of the recombinant cells was about 18.0-fold higher than in the extract from U. thermosphaericus strain A1. This made more rapid and simpler purification of the enzyme possible.

Figure 1

Multiple sequence alignment of meso-DAPDHs. The accession numbers of the aligned sequences are C. glutamicum ATCC13032 (YP_226858), B. sphaericus (BAB07799), Bacillus sp. B14905 (ZP_01724569), Cl. thermocellum ATCC27405 (ABN52156). Amino acid residues mutated for the creation of D-amino acid.

Figure 2

PAGE of purified meso-DAPDH from U. thermosphaeicus strain A1. A: Native PAGE of the purified enzyme. The purified enzyme was applied to a 7.5% acrylamide gel. The left and right lanes show the patterns of protein and activity staining, respectively. The triangle indicates the position of the bands. B: SDS-PAGE of the purified enzyme. The purified enzyme was applied to a SDS-PAGE on a 10% acrylamide gel. The left and right lanes show the positions of the molecular marker proteins and the purified enzyme.

Figure 3

Effect of temperature on the activity of NADP-dependent meso-DAPDH from U. thermosphaericus strain A1. After the enzyme (in 10 mM potassium phosphate buffer, pH 7.2) was incubated for 30 min at each temperature, the residual activity was determined using the standard assay at 50°C.