Nucleotide sequence and deduced amino acid sequence of Escherichia coli adenine phosphoribosyl-transferase and comparison with other analogous enzymes

ABSTRACT A new thermostable dipeptidase gene was cloned from the thermophile Brevibacillus borstelensis BCS-1 by genetic complementation of the d-Glu auxotroph Escherichia coli WM335 on a plate containing d-Ala-d-Glu. Nucleotide sequence analysis revealed that the gene included an open reading frame coding for a 307-amino-acid sequence with an M r of 35,000. The deduced amino acid sequence of the dipeptidase exhibited 52% similarity with the dipeptidase from Listeria monocytogenes. The enzyme was purified to homogeneity from recombinant E. coli WM335 harboring the dipeptidase gene from B. borstelensis BCS-1. Investigation of the enantioselectivity (E) to the P1 and P1′ site of Ala-Ala revealed that the ratio of the specificity constant (k cat /Km ) for l-enantioselectivity to the P1 site of Ala-Ala was 23.4 Ã¯Â¿Â½ 2.2 [E = (k cat /Km ) l,d /(k cat /Km ) d,d ], while the d-enantioselectivity to the P1′ site of Ala-Ala was 16.4 Ã¯Â¿Â½ 0.5 [E = (k cat /Km ) l,d /(k cat /Km ) l,l ] at 55Ã¯Â¿Â½C. The enzyme was stable up to 55Ã¯Â¿Â½C, and the optimal pH and temperature were 8.5 and 65Ã¯Â¿Â½C, respectively. The enzyme was able to hydrolyze l-Asp-d-Ala, l-Asp-d-AlaOMe, Z-d-Ala-d-AlaOBzl, and Z-l-Asp-d-AlaOBzl, yet it could not hydrolyze d-Ala-l-Asp, d-Ala-l-Ala, d-AlaNH2, and l-AlaNH2. The enzyme also exhibited β-lactamase activity similar to that of a human renal dipeptidase. The dipeptidase successfully synthesized the precursor of the dipeptide sweetener Z-l-Asp-d-AlaOBzl.

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The overexpression and complete amino acid sequence of Escherichia coli 3-dehydroquinase

Biochemical Journal ◽

10.1042/bj2380475 ◽

1986 ◽

Vol 238 (2) ◽

pp. 475-483 ◽

Cited By ~ 36

Author(s):

K Duncan ◽

S Chaudhuri ◽

M S Campbell ◽

J R Coggins

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Polypeptide Chain ◽

Amino Acid Residues ◽

Transcript Mapping ◽

Terminal Amino Acid ◽

Terminal Amino ◽

Terminal Amino Acid Sequence

The enzyme 3-dehydroquinase was purified in milligram quantities from an overproducing strain of Escherichia coli. The amino acid sequence was deduced from the nucleotide sequence of the aroD gene and confirmed by determining the amino acid composition of the overproduced enzyme and its N-terminal amino acid sequence. The complete polypeptide chain consists of 240 amino acid residues and has a calculated subunit Mr of 26,377. Transcript mapping revealed that aroD is a typical monocistronic gene.

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Molecular Cloning, Nucleotide Sequence, and Expression in Escherichia coli of a Hemolytic Toxin (Aerolysin) Gene from Aeromonas trota

Applied and Environmental Microbiology ◽

10.1128/aem.64.7.2473-2478.1998 ◽

1998 ◽

Vol 64 (7) ◽

pp. 2473-2478 ◽

Cited By ~ 19

Author(s):

Ashraf A. Khan ◽

Eungbin Kim ◽

Carl E. Cerniglia

Keyword(s):

Escherichia Coli ◽

Amino Acid ◽

Nucleotide Sequence ◽

Amino Acid Sequence ◽

Sequence Data ◽

Kanamycin Resistance ◽

Aeromonas Salmonicida ◽

Amino Acid Sequences ◽

Nucleotide Sequence Data ◽

Hemolytic Toxin

ABSTRACT Aeromonas trota AK2, which was derived from ATCC 49659 and produces the extracellular pore-forming hemolytic toxin aerolysin, was mutagenized with the transposon mini-Tn5Km1 to generate a hemolysin-deficient mutant, designated strain AK253. Southern blotting data indicated that an 8.7-kb NotI fragment of the genomic DNA of strain AK253 contained the kanamycin resistance gene of mini-Tn5Km1. The 8.7-kb NotI DNA fragment was cloned into the vector pGEM5Zf(−) by selecting for kanamycin resistance, and the resultant clone, pAK71, showed aerolysin activity in Escherichia coli JM109. The nucleotide sequence of the aerA gene, located on the 1.8-kbApaI-EcoRI fragment, was determined to consist of 1,479 bp and to have an ATG initiation codon and a TAA termination codon. An in vitro coupled transcription-translation analysis of the 1.8-kb region suggested that the aerA gene codes for a 54-kDa protein, in agreement with nucleotide sequence data. The deduced amino acid sequence of the aerA gene product ofA. trota exhibited 99% homology with the amino acid sequence of the aerA product of Aeromonas sobria AB3 and 57% homology with the amino acid sequences of the products of the aerA genes of Aeromonas salmonicida 17-2 and A. sobria 33.

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