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Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

ABSTRACT: Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were carefully studied, and we found that rhizavidin shares characteristics with bradavidin, streptavidin and avidin. However, we found that it is the first naturally occurring dimeric protein in the avidin protein family, in contrast with tetrameric (strept)avidin and bradavidin. Moreover, it possesses a proline residue after a flexible loop (GGSG) in a position close to Trp-110 in avidin, which is an important biotin-binding residue. [3H]Biotin dissociation and ITC (isothermal titration calorimetry) experiments showed dimeric rhizavidin to be a high-affinity biotin-binding protein. Its thermal stability was lower than that of avidin; although similar to streptavidin, it was insensitive to proteinase K. The immunological cross-reactivity of rhizavidin was tested with human serum samples obtained from cancer patients exposed to (strept)avidin. No significant cross-reactivity was observed. The biodistribution of the protein was studied by SPECT (single-photon emission computed tomography) imaging in rats. Similarly to avidin, rhizavidin was observed to accumulate rapidly, mainly in the liver. Evidently, rhizavidin could be used as a complement to (strept)avidin in (strept)avidin-biotin technology.

SUBMITTER: Helppolainen SH

PROVIDER: S-EPMC2267316 | biostudies-literature | 2007 Aug

REPOSITORIES: biostudies-literature

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Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

Helppolainen Satu H SH Nurminen Kirsi P KP Määttä Juha A E JA Halling Katrin K KK Slotte J Peter JP Huhtala Tuulia T Liimatainen Timo T Ylä-Herttuala Seppo S Airenne Kari J KJ Närvänen Ale A Jänis Janne J Vainiotalo Pirjo P Valjakka Jarkko J Kulomaa Markku S MS Nordlund Henri R HR

The Biochemical journal 20070801 3

Rhizobium etli CFN42 is a symbiotic nitrogen-fixing bacterium of the common bean Phaseolus vulgaris. The symbiotic plasmid p42d of R. etli comprises a gene encoding a putative (strept)avidin-like protein, named rhizavidin. The amino acid sequence identity of rhizavidin in relation to other known avidin-like proteins is 20-30%. The amino acid residues involved in the (strept)avidin-biotin interaction are well conserved in rhizavidin. The structural and functional properties of rhizavidin were car ...[more]

PMID: 17447892

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Project description:BackgroundThe chicken avidin gene family consists of avidin and several avidin related genes (AVRs). Of these gene products, avidin is the best characterized and is known for its extremely high affinity for D-biotin, a property that is utilized in numerous modern life science applications. Recently, the AVR genes have been expressed as recombinant proteins, which have shown different biotin-binding properties as compared to avidin.ResultsIn the present study, we have employed multiple biochemical methods to better understand the structure-function relationship of AVR proteins focusing on AVR2. Firstly, we have solved the high-resolution crystal structure of AVR2 in complex with a bound ligand, D-biotin. The AVR2 structure reveals an overall fold similar to the previously determined structures of avidin and AVR4. Major differences are seen, especially at the 1-3 subunit interface, which is stabilized mainly by polar interactions in the case of AVR2 but by hydrophobic interactions in the case of AVR4 and avidin, and in the vicinity of the biotin binding pocket. Secondly, mutagenesis, competitive dissociation analysis and differential scanning calorimetry were used to compare and study the biotin-binding properties as well as the thermal stability of AVRs and avidin. These analyses pinpointed the importance of residue 109 for biotin binding and stability of AVRs. The I109K mutation increased the biotin-binding affinity of AVR2, whereas the K109I mutation decreased the biotin-binding affinity of AVR4. Furthermore, the thermal stability of AVR2(I109K) increased in comparison to the wild-type protein and the K109I mutation led to a decrease in the thermal stability of AVR4.ConclusionAltogether, this study broadens our understanding of the structural features determining the ligand-binding affinities and stability as well as the molecular evolution within the protein family. This novel information can be applied to further develop and improve the tools already widely used in avidin-biotin technology.

Project description:Recently, Rhizobium etli, in addition to Agrobacterium spp., has emerged as a prokaryotic species whose genome encodes a functional machinery for DNA transfer to plant cells. To understand this R. etli-mediated genetic transformation, it would be useful to define how its vir genes respond to the host plants. Here, we explored the transcriptional activation of the vir genes contained on the R. etli p42a plasmid. Using a reporter construct harboring lacZ under the control of the R. etli virE promoter, we show that the signal phenolic molecule acetosyringone (AS) induces R. etli vir gene expression both in an R. etli background and in an Agrobacterium tumefaciens background. Furthermore, in both bacterial backgrounds, the p42a plasmid also promoted plant genetic transformation with a reporter transfer DNA (T-DNA). Importantly, the R. etli vir genes were transcriptionally activated by AS in a bacterial species-specific fashion in regard to the VirA/VirG signal sensor system, and this activation was induced by signals from the natural host species of this bacterium but not from nonhost plants. The early kinetics of transcriptional activation of the major vir genes of R. etli also revealed several features distinct from those known for A. tumefaciens: the expression of the virG gene reached saturation relatively quickly, and virB2, which in R. etli is located outside the virB operon, was expressed only at low levels and did not respond to AS. These differences in vir gene transcription may contribute to the lower efficiency of T-DNA transfer of R. etli p42a than of T-DNA transfer of pTiC58 of A. tumefaciensIMPORTANCE The region encoding homologs of Agrobacterium tumefaciens virulence genes in the Rhizobium etli CE3 p42a plasmid was the first endogenous virulence system encoded by the genome of a non-Agrobacterium species demonstrated to be functional in DNA transfer and stable integration into the plant cell genome. In this study, we explored the transcriptional regulation and induction of virulence genes in R. etli and show similarities to and differences from those of their A. tumefaciens counterparts, contributing to an understanding and a comparison of these two systems. Whereas most vir genes in R. etli follow an induction pattern similar to that of A. tumefaciens vir genes, a few significant differences may at least in part explain the variations in T-DNA transfer efficiency.

Project description:BackgroundA traditional concept in bacterial genetics states that housekeeping genes, those involved in basic metabolic functions needed for maintenance of the cell, are encoded in the chromosome, whereas genes required for dealing with challenging environmental conditions are located in plasmids. Exceptions to this rule have emerged from genomic sequence data of bacteria with multipartite genomes. The genome sequence of R. etli CFN42 predicts the presence of panC and panB genes clustered together on the 642 kb plasmid p42f and a second copy of panB on plasmid p42e. They encode putative pantothenate biosynthesis enzymes (pantoate-β-alanine ligase and 3-methyl-2-oxobutanoate hydroxymethyltransferase, respectively). Due to their ubiquitous distribution and relevance in the central metabolism of the cell, these genes are considered part of the core genome; thus, their occurrence in a plasmid is noteworthy. In this study we investigate the contribution of these genes to pantothenate biosynthesis, examine whether their presence in plasmids is a prevalent characteristic of the Rhizobiales with multipartite genomes, and assess the possibility that the panCB genes may have reached plasmids by horizontal gene transfer.ResultsAnalysis of mutants confirmed that the panC and panB genes located on plasmid p42f are indispensable for the synthesis of pantothenate. A screening of the location of panCB genes among members of the Rhizobiales showed that only R. etli and R. leguminosarum strains carry panCB genes in plasmids. The panCB phylogeny attested a common origin for chromosomal and plasmid-borne panCB sequences, suggesting that the R. etli and R. leguminosarum panCB genes are orthologs rather than xenologs. The panCB genes could not totally restore the ability of a strain cured of plasmid p42f to grow in minimal medium.ConclusionsThis study shows experimental evidence that core panCB genes located in plasmids of R. etli and R. leguminosarum are indispensable for the synthesis of pantothenate. The unusual presence of panCB genes in plasmids of Rhizobiales may be due to an intragenomic transfer from chromosome to plasmid. Plasmid p42f encodes other functions required for growth in minimal medium. Our results support the hypothesis of cooperation among different replicons for basic cellular functions in multipartite rhizobia genomes.

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Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

Publications

Rhizavidin from Rhizobium etli: the first natural dimer in the avidin protein family.

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