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- Cys-92, Cys-95, and the C-Terminal 12 Residues of the Vibrio harveyi Ferric Uptake Regulator (Fur) are Functionally Inessential
- Kun Sun , Shuang Cheng , Min Zhang , Fang Wang , Li Sun
- J. Microbiol. 2008;46(6):670-680. Published online December 24, 2008
- DOI: https://doi.org/10.1007/s12275-008-0113-3
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- 10 Scopus
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Abstract
- Ferric uptake regulator (Fur) is a global regulator involved in multiple aspects of bacterial life. The gene encoding the Vibrio harveyi Fur (FurVh) was cloned from a pathogenic V. harveyi strain isolated from diseased fish. FurVh shares 77% overall sequence identity with the Escherichia coli Fur (FurEc) and could complement a mutant of FurEc. Like FurEc, FurVh possesses two cysteine residues at positions 92 and 95, yet unlike FurEc, in which these cysteine residues constitute part of the metal ion coordination site and hence are vital to the repressor activity, C92 and C95 of FurVh proved to be functionally inessential. Further study identified a Vibrio Fur signature sequence, which is preserved in all the ten Vibrio Fur proteins that have been discovered to date but in none of the non-vibrio Fur proteins. Site-directed and random mutation analyses of the signature residues, the cysteine residues, and seven highly charged amino acid residues indicated that D9, H32, C137, and K138 of FurVh are functionally important but D9, C137, and K138 can be replaced by more than one functional substitutes. Systematic deletion analysis demonstrated that the C-terminal 12 residues of FurVh are functionally inessential. These results (i) indicated that the activation mechanism, or certain aspects of which, of FurVh is possibly different from that of FurEc; and (ii) suggested that it is not very likely that the C-terminal 12 residues play any significant role in the activation or stability of FurVh; and (iii) provided insights into the potential function of the local structure involving C137 and K138.
Journal Article
- NOTE] Bioluminescent Assay for Sphingolipid Ceramide N-Deacylase Using Vibrio harveyi Dark Mutant M-17
- Ki Woong Cho
- J. Microbiol. 2008;46(5):585-589. Published online October 31, 2008
- DOI: https://doi.org/10.1007/s12275-008-0114-2
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- 1 Scopus
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Abstract
- A new bioluminescent assay method for the activity of sphingolipid ceramide N-deacylase (SCDase: EC3.5.1.69) as well as ceramidase (CDase: EC 3.5.1.23) was developed using bioluminescent marine bacteria. Enzymatically synthesized ceramide (N-myristoyl sphigosine, C14:0-18:1) and commercial SCDase were used in this demonstration, and myristic (tetradecanoic, C14:0) acid produced by the SCDase hydrolysis was quantified using Vibrio harveyi M-17, a dark mutant of V. harveyi. The in vivo light intensity of M-17 was stimulated up to thousands fold in the presence of myristic acid, was used for this assay. SCDase activity with as little as 10 μU and 5 nM of myristic acid production were detected in less than one min. <br>The assay worked well for the determination of Km and chromatographic fraction assay.
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