Purification, characterization, and gene cloning of one subunit of the heteromeric transcriptional activator, MvaT, of the Pseudomonas mevaloniimvaAB operon

Richard Scott Rosenthal, Purdue University

Abstract

Regulation of the Pseudomonas mevalonii mvaAB operon that encodes HMG-CoA reductase (E.C. 1.1.1.88) and HMG-CoA lyase (E.C. 4.1.3.4) occurs at the level of transcription. The gene products of the mvaAB operon facilitate the first two steps in the catabolism of mevalonate. MvaT, the transcriptional activator of the mvaAB operon, has been purified over 1,200-fold and partially characterized. MvaT is expressed constitutively and binds in vitro to the regulatory cis-element upstream of the mvaAB operon with a K$\rm\sb{d} \sb{app}$ of 2 nM. Mevalonate is necessary for activation of transcription in vivo, however, in vitro binding of MvaT to the cis-element occurs in the absence of mevalonate. Purification of MvaT enriched for 2 dissimilar polypeptides of approximate molecular mass 15 and 16 kDa. These polypeptides were termed P15 and P16, respectively. P15, P16, and MvaT activity comigrated during extensive purification that included DNA-affinity and size exclusion chromatography, and sucrose density gradient centrifugation. P15 and P16 also comigrated when purified MvaT was subjected to denaturing isoelectric focusing. Treatment of purified MvaT with the crosslinking reagent dimethylsuberimidate resulted in the formation of a 31 kDa polypeptide complex that contained amino acid sequence from P15 and P16. The apparent association of P15 and P26 in solution and their comigration with MvaT suggests that MvaT is a heteromeric protein composed of P15 and P16 subunits. The molecular mass of MvaT, estimated by size exclusion chromatography, was approximately 33 kDa, a value consistent with MvaT being a dimeric protein composed of P15 and P16. A portion of the gene that encodes P16 was cloned from P. mevlonii using a polymerase chain reaction approach that utilized degenerated PCR primers directed against the N-termini of P15 and P16. The deduced amino acid sequence of P16 predicts a polypeptide having a molecular weight of 14,300 and estimated by computer software to have a pI of approximately 10. As judged by codon frequency, codon usage in the P16 gene is essentially identical to that of mvaA and mvaB.

Degree

Ph.D.

Advisors

Rodwell, Purdue University.

Subject Area

Biochemistry|Molecular biology|Microbiology

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