Nucleotide Sequencing of Phenylalanine Dehydrogenase Gene fromBacillus badiusIAM 11059

Abstract

I ,9) is more stable and shows a wider specificity which facilitates the synthesis of varieties of L-amino acids from a-keto acids by the reductive amination reaction.4 ) The nucleotide sequences for the pdh genes from B. sphaericus,9) Thermoactinomyces intermedius, 10) and Rhodococcus Sp.I1) were published. For this paper, we sequenced the pdh gene from B. badius to study the structural relationship from other proteins.The growth condition of B. badius and the cloning of the pdh gene were described previously.2) Plasmids were purified by a plasmid purification kit Qiagen (Qiagen Inc., U.S.A.).The enzyme was purified from B. badius lAM 11059 and Escherichia coli JM109/pBBI 2 ) to homogeneity with a procedure involving ammonium sulfate fractionation, DEAE-Toyopearl, Butyl-Toyopearl, Sephadex G-200, Mono-Q, and Phenyl-Superose HR5/5 column chromatographies. Before the analysis of the NHz-terminal amino acid sequence, the enzyme sample was passed through a TSK Phenyl-5PW column (0.75 x 7.5 em, Tosoh Corp., Japan), and fractionated with a linear gradient of 20-S0% (v/v) acetonitrile containing 0.05% (v/v) trifiuoroacetic acid. The purified PheDH from B. badius (l.Omg (27.SnM» was digested with lysyl-end peptidase (4.2~g (0.14nM), Wako Pure Chemicals, Japan) at 30°C for 16 h. The resulting peptides were separated by HPLC (Waters 600E, U.S.A.), with a reversed-phase column (To soh Corp, Japan, TSK gel ODS80Ts, ¢46 x 150mm). The amino acid sequences of the peptides and that of N-terminus region were identified with an automatic protein sequencer 6625 (MilliGen, U.S.A.).The fragment to be sequenced was subcloned to a plasmid vector pBluescript. A kilo-sequencing kit (Takara Shuzo, Japan) was used to generate shorter clones suitable for sequencing. The sequencing was done by the dideoxy chain termination procedure of Sanger et al., using an automatic DNA sequencer ALF (Pharmacia, Sweden). It was also done with [a-35 S}dCTP as the radioactive label. A Sphl-Clal fragment (1.6 kb) containing the entire pdh gene based on the expression of the enzyme activity, was prepared from the recombinant plasmid pBB 1 2 ) and used for sequence analysis. The nucleotide sequence of the 1.6 kb Sphl-Clal fragment (given in the data base t) showed that there is an ORF encoding the 1140 bp pdh gene ( Fig.) specifying a protein of a calculated molecular weight of 41,350, which is in excellent agreement with the reported value of 41,000 to 42,000 by SDS-PAGE. 2 ) We have purified the enzyme from both of B. badius and the E. coli JMI09 transformant and found that the N-terminal amino acid sequences are identical. The amino acid sequences of some of internal peptides were in good agreement with the predicted amino acid sequence as indicated in Computer alignments by the SWISS-PROT and NBRF-PIR data bases were made by the DNASIS program (Hitachi, Japan). The deduced primary structure of B. badius PheDH is similar to PheDH from B. sphaericus (67.9% identical over 377 a.a.)9) and PheDH from T. intermedius (56.6% over 346 a.a.), 1 0) leucine dehydro...

Crystallization of NAD+-dependent phenylalanine dehydrogenase fromNocardia sp239

“…Crystals were grown by the hanging-drop method of vapour diffusion using Linbro crystallization plates by mixing 5 pl of (Okazaki et al, 1988); Bacillus badius PheDH (Bh ) (Yamada et al, 1995); Thermoactinomvces intermedius PheDH ( Ti) ; Rhodococcus M4 PheDH (RhM4) (Brunhuber et al, 1994); Bacillus stearothermophilus LeuDH (Bst) (Nagata et al, 1988) and Clostridium svmbiosum GluDH (Cs) (Teller et al, 1992). The amino acids highlighted in bold are those which are identical across the superfamily of amino-acid dehydrogenases.…”

Kinetic analysis of phenylalanine dehydrogenase mutants designed for aliphatic amino acid dehydrogenase activity with guidance from homology‐based modelling

“…These are unchanged in LeuDH, but in PheDH leucine replaces valine in the first position, and the replacement of an alanine residue with glycine in the second position, on the opposite side of the binding pocket, is thought to create a deeper binding pocket for access of substrates with large aromatic side-chains. More recent sequence determinations support this proposal, with LeuDH from Thermoactinomyces intermedius and Bacillus cereus [13,14], and valine dehydrogenase (ValDH) [15][16][17], another member of the family that accepts aliphatic amino acid substrates, all having valine and alanine at the respective positions, whereas leucine and glycine are present in the PheDH from B. badius [18,19] and Sporosarcina ureae [20] (Fig. 1).…”

Crystal structure of NAD+-dependent Peptoniphilus asaccharolyticus glutamate dehydrogenase reveals determinants of cofactor specificity

“…GDH belongs to the amino acid dehydrogenase enzyme superfamily, members of which members display differential substrate specificity and include valine, leucine and phenylalanine dehydrogenases (Britton et al, 1993). This enzyme superfamily has considerable potential in the production of novel non-proteinogenic amino acids for the pharmaceutical industry (Paradisi et al, 2007;Yamada et al, 1995). Phenylalanine dehydrogenase has also been widely exploited for diagnosis of phenylketonuria (Nakamura et al, 1996) and glutamate dehydrogenase similarly for measurement of .…”