Kinetics of Phosphomevalonate Kinase from <i>Saccharomyces cerevisiae</i>

<div><p>The mevalonate-based isoprenoid biosynthetic pathway is responsible for producing cholesterol in humans and is used commercially to produce drugs, chemicals, and fuels. Heterologous expression of this pathway in <i>Escherichia coli</i> has enabled high-level production of the antimalarial drug artemisinin and the proposed biofuel bisabolane. Understanding the kinetics of the enzymes in the biosynthetic pathway is critical to optimize the pathway for high flux. We have characterized the kinetic parameters of phosphomevalonate kinase (PMK, EC 2.7.4.2) from <i>Saccharomyces cerevisiae</i>, a previously unstudied enzyme. An <i>E. coli</i> codon-optimized version of the <i>S. cerevisiae</i> gene was cloned into pET-52b+, then the C-terminal 6X His-tagged protein was expressed in <i>E. coli</i> BL21(DE3) and purified on a Ni²⁺ column. The K_M of the ATP binding site was determined to be 98.3 µM at 30°C, the optimal growth temperature for <i>S. cerevisiae</i>, and 74.3 µM at 37°C, the optimal growth temperature for <i>E. coli</i>. The K_M of the mevalonate-5-phosphate binding site was determined to be 885 µM at 30°C and 880 µM at 37°C. The V_max was determined to be 4.51 µmol/min/mg enzyme at 30°C and 5.33 µmol/min/mg enzyme at 37°C. PMK is Mg²⁺ dependent, with maximal activity achieved at concentrations of 10 mM or greater. Maximum activity was observed at pH = 7.2. PMK was not found to be substrate inhibited, nor feedback inhibited by FPP at concentrations up to 10 µM FPP.</p></div

Initial reaction velocity as a function of mevalonate-5-phosphate concentration.

<p>Closed circles are data for incubation at 37°C and open circles are data for incubation at 30°C.</p

Divalent cation dependence.

<p>Closed circles are data for Mg²⁺ and open circles are data for Mn²⁺.</p

Functional Characterization of the Origin of Replication of pRN1 from <i>Sulfolobus islandicus</i> REN1H1

<div><p>Plasmid pRN1 from <i>Sulfolobus islandicus REN1H1</i> is believed to replicate by a rolling circle mechanism but its origin and mechanism of replication are not well understood. We sought to create minimal expression vectors based on pRN1 that would be useful for heterologous gene expression in <i>S. acidocaldarius</i>, and in the process improve our understanding of the mechanism of replication. We constructed and transformed shuttle vectors that harbored different contiguous stretches of DNA from pRN1 into <i>S. acidocaldarius E4-39</i>, a uracil auxotroph. A 232-bp region 3’ of <i>orf904</i> was found to be critical for pRN1 replication and is therefore proposed to be the putative origin of replication. This 232-bp region contains a 100-bp stem-loop structure believed to be the double-strand origin of replication. The loop of the 100-bp structure contains a GTG tri-nucleotide motif, a feature that was previously reported to be important for the primase activity of Orf904. This putative origin and the associated <i>orf56</i> and <i>orf904</i> were identified as the minimal replicon of pRN1 because transformants of plasmids lacking any of these three features were not recovered. Plasmids lacking <i>orf904</i> and <i>orf56</i> but harboring the putative origin were transformable when <i>orf904</i> and <i>orf56</i> were provided <i>in-trans</i>; a 75-bp region 5’ of the <i>orf904</i> start codon was found to be essential for this complementation. Detailed knowledge of the pRN1 origin of replication will broaden the application of the plasmid as a genetic tool for <i>Sulfolobus</i> species. </p> </div

Construction of pRN1-based shuttle vectors.

<p>(A) Map of pRN1 showing native genes. (B) Map of pRN1-based shuttle vector(s) showing the 232-bp putative pRN1 origin of replication (red double arrow). Each vector harbors the pUC19 Ap^r gene and origin (red box), <i>pyrE</i> with or without <i>lacS</i> and a segment of pRN1 highlighted by black arrows. The <i>lacS</i> gene was deleted from pRSP1, 3, and 5 to generate pRSP2, 4, and 6. </p

Extraction plasmids from <i>S. acidocaldarius</i> transformants and re-transformation into <i>E. coli</i>.

<p>(A) pRN1-based shuttle vectors were recovered from <i>S. acidocaldarius</i> transformants and re-transformed into <i>E. coli</i> to verify that the plasmids were self-replicating. The identity of the plasmids was confirmed by their EcoRI digestion finger-print. (A) EcoRI digestion finger-print of pRSP1, 5 and 7 recovered from (A) <i>S. acidocaldarius </i><i>E4-39</i> transformants and (B) <i>E. coli</i> re-transformed with plasmid DNA from the S. <i>acidocaldarius</i> transformants analyzed in (A).</p

Stem-loop structures identified within the 232-bp pRN1 putative origin of replication.

<p>(A) The secondary structure of 232-bp putative origin of replication determined using DNA mFold, revealed the presence of 100-bp (4734 - 4833) and 19-bp (4693 - 4711) stem-loop (SL) structures at 70°C. (B) DNA sequence of the 232-bp putative origin of replication showing the 100-bp SL (single underline) and 19-bp SL (double underline) in pRN1 origin. Deletions made in pRSP1a (between ∆), pRSP1b (between∇), pRSP1c (between ▼) and pRSP1d (between ▲) are highlighted by the triangles, while the deletion in pRSP1e is shaded. The ‘GTG’ motif (box) and the loop of the 100-bp SL (red) are also highlighted. </p

<i>In-trans</i> complementation of <i>orf56</i> and <i>orf904</i> deletion mutants of pRSP1.

<p>(A) Deletion mutants of pRSP1 generated by deleting <i>orf904</i> (pRSP9) and <i>orf56</i> (pRSP10/pRSP10b) from pRSP1; pRSP10 contained a complete <i>orf904</i> under the control of <i>orf56</i> transcription and translation elements, while pRSP10b contained an additional 75-bp at the 5’ of <i>orf904</i> start codon. Each plasmid contains pUC19 and pRN1 origins of replication, the <i>pyrE-lacS</i> cassette transcribed by the S. <i>solfataricus </i><i>thsB</i> promoter [P(β)] and <i>lacS</i> terminator element [lacS(T)], and AP^r gene (ampicillin resistance). (B) <i>Cis</i>-acting elements at the 5’ of <i>orf56</i> and <i>orf904</i> in each plasmid. Transcription of <i>orf904</i> in pRSP10 and pRSP10b is controlled by of <i>orf56</i> promoter elements (big bold box) with putative TATA box A (bold single underline) and TATA box B (single underline). The 75-bp element 5’of <i>orf904</i> in pRSP10b (bold underline) contains a putative RBS (small box). The start of <i>orf904</i> is bolded, and the stop of <i>orf56</i> is represented by (***). </p

Strategy of mapping pRN1 putative origin of replication (double red arrow in Figure 1).

<p>(A) Various deletions were made within the ‘putative ori’ in pRSP1 to generate plasmids highlighted in each rectangular box to determine the role of the deleted regions in pRN1 replication. We constructed pRSP1-NO and pRSP3-NO by deleting 232-bp and 207-bp, respectively, from the putative origin. (B) Three-nucleotide deletion mutants of pRSP1 were constructed to map the loop of stem-loop structure within pRN1 origin. Each construct was transformed into <i>S. acidocaldarius</i> and scored for production of LacS expressing transformants. </p