11/29/2011 1 The dialkylglycine decarboxylase repressor DgdR. Functional aspects and relation to other LysR proteins 1. Dialkylglycine amino acids 2. In vivo and in vitro studies on the DgdR repressor protein 3. Phylogenetic analysis of DgdR and DgdA proteins 4. Recent x-ray crystal structures of tetrameric LysR proteins
11/29/2011 2, -Dialkylglycines: -aminoisobutyric acid (Aib) R- and S-isovaline (Iva) D(R)- and L(S)-alanine
11/29/2011 3 Aminoisobutyrate (Aib) and Isovaline (Iva) occurrence 1. Fungal peptaibol antibiotic peptides Alamethicin: Ac-Aib-Pro-Aib-Ala-Aib-Ala-Gln-Aib-Val-Aib-Gly-Leu-Aib-Pro-Val-Aib-Aib-Glu-Gln-PheOH Cephaibol A: Ac-Phe-Aib-Aib-Aib-Aib-Gly-Leu-(R) Iva-Aib-Pro-Gln--(R) Iva-Pro-Aib-Pro-PheOH Bunkoczi, G. et al., J. Pept. Sci (2003) 9: 745. Non-ribosomal peptide synthase. Biosynthesis route to Aib and Iva is unknown.
11/29/2011 4 Aminoisobutyrate (Aib) and Isovaline (Iva) occurrence 2. Meteorites and bolide impact zones. Jeff Bada, Scripps Glavin & Dworkin PNAS (2009) 106, 5487.
11/29/2011 5 Burkholderia cepacia and several other bacteria and fungi (but not E. coli) can utilize Aib as the sole N source. The dialkylglycine decarboxylase was first isolated, purified, and characterized in the 60s by Esmond Snell and others. Dgd genes were cloned and sequenced at UAF (J. Biol. Chem. (1990) 265: 5531). by inserting B. cepacia DNA into an E. coli plasmid, and screening the recombinants with Aib/glucose agar. dgdr, the dialkylglycine repressor structural gene dgda, the dialkylglycine decarboxylase structural gene
11/29/2011 6 Basis for commercial interest and U.S. patents to JK and UA: 1. Aib is actively transported into many cell types, is non-toxic, and there are no known eukaryotic dgd gene analogues. 2. The E. coli lac repressor-operator system is functional in mouse cells.
11/29/2011 7 The biological role of the dgd genes and gene products is still not entirely clear. Dialkylglycine decarboxylase can process several amino acids including Aib, R-isovaline, D(R)-alanine, and others. So which one is the natural substrate?
11/29/2011 8 The natural substrate would be a good substrate, and also induce expression of the decarboxylase structural gene. These genes would have evolved together to be turned on by, and process, an amino acid. pror pros none H H H CH 3 No decarboxylation H CH 2 CH 3 H CH 2 CH 2 CH 3 H CH(CH 3 ) 2 H CH(CH 3 )CH 2 CH 3 CH 3 H Decarb, but no induction CH 3 CH 2 H CH 3 CH 2 CH 2 H (CH 3 ) 2 CH H CH 3 CH 2 CH(CH 3 ) H Rapid decarboxylation V. Slow decarboxyl n CH 3 CH 3 CH 3 CH 2 CH 3 CH 3 CH 2 CH 2 CH 3 Decarb, but no induction CH 3 CH 2 CH 3 E. coli JM109/pGEM7Z14: 20 h growth in YT medium + 20 mm amino acid. CH 3 CH 2 CH 2 CH 3 V. Slow decarboxyl n -CH 2 CH 2 CH 2 CH 2 - Decarboxylase specific activity (u/mg)
11/29/2011 9 Conclusion: the dgd genes evolved to metabolize Aib S-isovaline induces dgda expression (but R-isovaline is the better substrate for the dialkylglycine decarboxylase).
11/29/2011 10 The dgdr gene was overexpressed in E. coli and purified by standard methods. As has been found for many LysR-type DNA binding proteins, the DgdR protein can be concentrated only in the presence of high-salt buffers.
11/29/2011 11 Intergenic region and 5 -ends of dgd genes dgda dgda transcript dgdr -10-35 Bacterial RNA polymerase promoter Footprint of DgdR protein This section of DNA was used in gel shift assays to measure effects of various amino acid on DgdR-DNA binding.
11/29/2011 12 Gel shift assay of purified DgdR protein + 594-bp DNA including 5 -ends of dgda and dgdr genes. fast (f) slow (s) DNA is bent and more rigid. This half of the PAGE gel contains 10 mm Aib
11/29/2011 13 The same four amino acids induce dgda gene expression in vivo, and enhance the formation of slow-shifted protein-dna complexes in vitro. In vivo inducers Other amino acids have no effect on dgda gene expression, or on formation of slow-shifted protein- DNA complexes. In vivo non-inducers amino acid This is a structure-activity correlation that implicates the formation of the slow-moving (bent) DgdR-DNA complex as a necessary component in expression of the dgda gene.
11/29/2011 14 The DgdR experimental work was carried out by graduate students Lilly Allen and HongHong Sun, with help from undergraduates Susan Bray, Marcia O Brien, and Renee Stapleton.
11/29/2011 15 Sequence analysis of DgdR homologues
11/29/2011 16 DgdR DgdA All dgda homologue genes have upstream ORFs dgdr homologues- that that are strongly similar to the LysR family. tblastn using as a probe the B. cepacia DgdA protein sequence DgdA hits in genbank database Now look for open reading frames upstream of and divergent from the dgda gene homologues.
11/29/2011 17 13 top BLAST hits for B. cepacia DgdA decarboxylase protein Active site catalytic Gln, which differentiates authentic dialkylglycine decarboxylases from other vitamin B6- dependent enzymes. clustalx.2
11/29/2011 18 Clustal alignment of DgdR proteins encoded by genes adjacent to the same decarboxylase-encoding genes (part of) the regulatory domain expected to bind Aib
11/29/2011 19 The phylogenetic trees derived from sequence alignments of two Dgd protein groups are virtually identical. This suggests that each gene pair has remained together and undergone evolutionary change independent of other dgd gene pairs.
11/29/2011 20 Therefore, the dgd regulator-enzyme gene pairs have probably existed for millions of years, providing an essential biological or ecological function for various microorganisms. But what is that function??
11/29/2011 21 About 20% of bacterial regulatory genes and proteins are in the LysR family. About 280 10 aa long. Contain a Winged helix-turn-helix DNA binding domain at the N-terminus A long linker helix connecting the DBD to the C-terminus An / regulatory, or effector binding, domain consisting of two halves linked by two beta strands. A C-terminal helix that may be required for DNA binding
11/29/2011 22 X-ray crystal structures Most are regulatory domain only (~20 in protein data bank) First was CysB with sulfate ion bound (Tyrrell, R et al Structure 5, 1017-1032) Full-length: (November 2011) Vibrio cholerae virulence activator, AphB (3SZP.pdb) Arginine permease ArgP from Mycobacterium tuberculosis (3ISP.pdb) Toluenesulfonic acid LysR-type regulator TsaR from Comamonas testosteroni (3FXQ.pdb) CbnR
11/29/2011 23 Two (or three) domains: winged-helix-turn-helix; / regulatory domain, which includes the C-terminal helix.
11/29/2011 24 All structures show a dimer-of-dimers quaternary structure. The monomers in each dimer adopt two conformations: extended and compact. The difference is how the DBD is situated relative to the RD: the connecting helix is either long, or bent back onto the RD.
11/29/2011 25 CbnR Muraoka et al. J. Mol. Biol. 2003
11/29/2011 26 The N-terminal w-hth domains present a linear curved surface (to DNA?).
11/29/2011 27 CbnR Muraoka et al. J. Mol. Biol. 2003
11/29/2011 28 Hypothesis by Monferrer et al for conformation change ( Mol Micro 2010) Sliding dimer hypothesis by Porrúa et al (Mol. Micro. 2007)
11/29/2011 29 Un-induced CbnR Induced TsaR
11/29/2011 30 Clustal alignment of DgdR proteins encoded by genes adjacent to a decarboxylase-encoding gene Where do these conserved aa s map onto the known LysR regulatory domain structures? Adjacent to the effector binding site?
11/29/2011 31 The End