Purification and characterization of the heteromeric transcriptional activator MvaT of the Pseudomonas mevalonii mvaAB operon.
The mvaAB operon of Pseudomonas mevalonii encodes HMG-CoA reductase (EC 1.1.1.88) and HMG-CoA lyase (EC 4.1.3.4), enzymes that catalyze the initial reactions of mevalonate catabolism in this organism. Expression of this operon is regulated by the constitutively expressed transcriptional activator protein MvaT that binds in vitro to an upstream regulatory element. Mevalonate is essential for activation of transcription in vivo, and in vitro data demonstrated that MvaT binds to the mvaAB cis-regulatory element in the absence of mevalonate with a Kd,app of 2 nM. Purification of MvaT enriched for two polypeptides of approximate molecular mass 15 kDa and 16 kDa, designated P15 and P16. MvaT, assayed by its DNA-binding activity, comigrated with P15 and P16 during DNA-affinity chromatography, size-exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated during denaturing isoelectric focusing of purified MvaT. Treatment of MvaT with dimethylsuberimidate formed a 31-kDa polypeptide complex that contained N-terminal sequences from P15 and P16. The apparent association of P15 and P16 in solution and their copurification with MvaT activity strongly suggests that MvaT is comprised of these two subunits. Size-exclusion chromatography gave an estimated molecular mass for MvaT of 33 kDa. A partial DNA sequence of the P16 gene was obtained using PCR employing degenerate primers directed against the N-termini of P15 and P16. P16 appears to be comprised of at least 128 aminoacyl residues having a predicted molecular mass of 14.3 kDa.
Rosenthal
R S
RS
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
Rodwell
V W
VW
D000595:Amino Acid Sequence; D001426:Bacterial Proteins; D001483:Base Sequence; D003001:Cloning, Molecular; D003432:Cross-Linking Reagents / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000737:chemistry; D019281:Dimerization; D004120:Dimethyl Suberimidate / Q000378:metabolism; D015964:Gene Expression Regulation, Bacterial / Q000235:genetics; D006903:Hydroxymethylglutaryl CoA Reductases / Q000235:genetics; D008798:Mevalonic Acid / Q000378:metabolism; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009876:Operon / Q000235:genetics*; D007652:Oxo-Acid-Lyases / Q000235:genetics; D011549:Pseudomonas / Q000201:enzymology*; D017422:Sequence Analysis, DNA; D015534:Trans-Activators / Q000737:chemistry*; D014157:Transcription Factors / Q000737:chemistry*; D015533:Transcriptional Activation / Q000502:physiology*
D001426:Bacterial Proteins; D003432:Cross-Linking Reagents; D004268:DNA-Binding Proteins; C111692:MvaT protein, Pseudomonas; D015534:Trans-Activators; D014157:Transcription Factors; D004120:Dimethyl Suberimidate; D006903:Hydroxymethylglutaryl CoA Reductases; D007652:Oxo-Acid-Lyases; C021410:3-hydroxy-3-methylglutaryl-coenzyme A lyase; D008798:Mevalonic Acid