AAP, Aminopeptidase from Vibrio Proteolyticus (Aeromonas Proteolytica)

A halotolerant bacterium was isolated from a saline lake located in Mallorca, Spain. Cells of the strain, designated MACL01T, were Gram-negative, rod-shaped and motile by means of polar flagella. Colonies of strain MACL01T were white to cream in TSA medium, turning brown after 7 days of incubation; they were blue in thiosulphate/citrate/bile salts/sucrose agar medium. A neighbour-joining phylogenetic analysis based on 16S rRNA gene sequences showed that strain MACL01T belongs to the genus Photobacterium, in which it forms a distinct lineage together with Photobacterium rosenbergii and Photobacterium ganghwense (showing 96.9 and 96.2 % similarity, respectively). The most closely related taxon according to phylogenetic analysis of the rpoA gene is also P. rosenbergii (90 % similarity). The recA gene also showed low similarity (83.7, 83.4 and 82.4 %, respectively) with respect to those of Vibrio proteolyticus LMG 3772T, Photobacterium leiognathii LMG 4228T and P. rosenbergii LMG 22223T. Neighbour-joining phylogenetic analysis of the rpoA and recA genes confirms that strain MACL01T belongs to the genus Photobacterium, forming a branch together with P. rosenbergii. Strain MACL01T was able to grow in 0–8 % NaCl. Growth occurred between 4 and 37 °C (optimum, 28 °C) and at pH 5–8.5. Luminescence was negative on marine agar. Strain MACL01T was found to be sensitive to the vibriostatic agent O/129. It reduced nitrate to nitrite, produced β-galactosidase and hydrolysed gelatin, but did not produce arginine dihydrolase, indole or acetoin. Strain MACL01T used several carbohydrates and fermented glucose, l-arabinose and sucrose. The most abundant fatty acids were summed feature 3 (32.6 %; comprising C16 : 1 ω7c and/or C15 : 0 iso 2-OH), C16 : 0 (21.2 %) and C18 : 1 ω7c (19.9 %). The G+C content of the genomic DNA was 49.8 mol%. On the basis of genotypic, phenotypic, chemotaxonomic and phylogenetic results, strain MACL01T (=LMG 22194T=CECT 5860T) should be classified as the type strain of a novel species of the genus Photobacterium, for which the name Photobacterium halotolerans sp. nov. is proposed.

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Function of the N-terminal propeptide of an aminopeptidase from Vibrio proteolyticus

Biochemical Journal ◽

10.1042/bj3500671 ◽

2000 ◽

Vol 350 (3) ◽

pp. 671-676 ◽

Cited By ~ 11

Author(s):

Zhen-Zhong ZHANG ◽

Satoru NIRASAWA ◽

Yoshiaki NAKAJIMA ◽

Michiteru YOSHIDA ◽

Kiyoshi HAYASHI

Keyword(s):

Escherichia Coli ◽

Enzyme Activity ◽

Active Region ◽

Protein Expression ◽

Inclusion Bodies ◽

Active Enzyme ◽

Vibrio Proteolyticus ◽

Show Evidence ◽

Inhibited Enzyme

An aminopeptidase from Vibrio proteolyticus was translated as a preproprotein consisting of four domains: a signal peptide, an N-terminal propeptide, a mature region and a C-terminal propeptide. Protein expression and analysis of the activity results demonstrated that the N-terminal propeptide was essential to the formation of the active enzyme in Escherichia coli. Urea dissolution of inclusion bodies and dialysis indicated that the N-terminal propeptide could facilitate the correct folding of the enzyme in vitro. Using l-Leu-p-nitroanilide as the substrate, the kinetic parameters (kcat and Km) of the pro-aminopeptidase and processed aminopeptidases were analysed. The results suggested that the N-terminal propeptide inhibited enzyme activity of the mature region. In contrast, the C-terminal propeptide did not show evidence of forming an active enzyme, of correctly folding in vitro or of inhibiting the active region.

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Chitobiose Phosphorylase from Vibrio proteolyticus, a Member of Glycosyl Transferase Family 36, Has a Clan GH-L-like (α/α)6 Barrel Fold

Structure ◽

10.1016/j.str.2004.03.027 ◽

2004 ◽

Vol 12 (6) ◽

pp. 937-947 ◽

Cited By ~ 78

Author(s):

Masafumi Hidaka ◽

Yuji Honda ◽

Motomitsu Kitaoka ◽

Satoru Nirasawa ◽

Kiyoshi Hayashi ◽

...

Keyword(s):

Glycosyl Transferase ◽

Vibrio Proteolyticus

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Nomenclature Abstract for Vibrio proteolyticus (Merkel et al. 1964) Baumann et al. 1982.

The NamesforLife Abstracts ◽

10.1601/nm.2999 ◽

2003 ◽

Author(s):

Charles Thomas Parker ◽

Sarah Wigley ◽

George M Garrity

Keyword(s):

Vibrio Proteolyticus

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Reaction mechanism of chitobiose phosphorylase from Vibrio proteolyticus: identification of family 36 glycosyltransferase in Vibrio

Biochemical Journal ◽

10.1042/bj20031171 ◽

2004 ◽

Vol 377 (1) ◽

pp. 225-232 ◽

Cited By ~ 37

Author(s):

Yuji HONDA ◽

Motomitsu KITAOKA ◽

Kiyoshi HAYASHI

Keyword(s):

Reaction Mechanism ◽

Amino Acid ◽

Amino Acid Sequence ◽

Substrate Inhibition ◽

Acceptor Substrate ◽

Vibrio Proteolyticus ◽

Cellobiose Phosphorylase ◽

Synthetic Reaction ◽

Vibrio Furnissii ◽

High Degree

A family 36 glycosyltransferase gene was cloned from Vibrio proteolyticus. The deduced amino acid sequence showed a high degree of identity with ChBP (chitobiose phosphorylase) from another species, Vibrio furnissii. The recombinant enzyme catalysed the reversible phosphorolysis of (GlcNAc)2 (chitobiose) to form 2-acetamide-2-deoxy-α-d-glucose 1-phosphate [GlcNAc-1-P] and GlcNAc, but showed no activity on cellobiose, indicating that the enzyme was ChBP, not cellobiose phosphorylase. In the synthetic reaction, the ChBP was active with α-d-glucose 1-phosphate as the donor substrate as well as GlcNAc-1-P to produce β-d-glucosyl-(1→4)-2-acetamide-2-deoxy-d-glucose with GlcNAc as the acceptor substrate. The enzyme allowed aryl-β-glycosides of GlcNAc as the acceptor substrate with 10–20% activities of GlcNAc. Kinetic parameters of (GlcNAc)2 in the phosphorolysis and GlcNAc-1-P in the synthetic reaction were determined as follows: phosphorolysis, k0=5.5 s−1, Km=2.0 mM; synthetic reaction, k0=10 s−1, Km=14 mM, respectively. The mechanism of the phosphorolytic reaction followed a sequential Bi Bi mechanism, as frequently observed with cellobiose phosphorylases. Substrate inhibition by GlcNAc was observed in the synthetic reaction. The enzyme was considered a unique biocatalyst for glycosidation.

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