Yeast protein farnesyltransferase. Binding of S-alkyl peptides and related analogues

[reaction: see text] Protein farnesyltransferase (PFTase) catalyzes alkylation of cysteine residues by farnesyl diphosphate (FPP). The dissociation constants for the PFTase-peptide analogue complexes for the series of analogues fl-RTRC(X)VIA (X = H, methyl, dodecyl, farnesyl) were measured by fluorescence anisotropy. The results indicate that an ionizable sulfhydryl moiety is important for substrate binding and the farnesyl group in the product facilitates binding.     

Recombinant squalene synthase. Synthesis of non-head-to-tail isoprenoids in the absence of NADPH

Squalene synthase (SQase) catalyzes two consecutive reactions in sterol biosynthesis. The first is the condensation of two molecules of farnesyl diphosphate (FPP) to form a cyclopropylcarbinyl intermediate, presqualene diphosphate (PSPP). The subsequent conversion of PSPP to squalene (SQ) involves an extensive rearrangement of the carbon skeleton and a NADPH-dependent reduction. Incubation of a truncated soluble form of recombinant yeast SQase with FPP in buffer lacking NADPH gave (1R,2R,3R)-PSPP. As the incubation continued, SQase catalyzed the subsequent conversion of PSPP to a mixture of triterpenes. Two of the major products, (Z)-dehydrosqualene (DSQ) and (R)-12-hydroxysqualene (HSQ), have the same 1'-1 linkage between the farnesyl units from FPP that is found in squalene. The other major product, (10S,13S)-10-hydroxybotryococcene (HBO), has a 1'-3 linkage between the farnesyl units. Small quantities of (S)-HSQ and (10R,13S)-HBO were also formed. Three additional triterpenes, the allylic isomers of HSQ and HBO, and an unidentified alcohol were produced in minor amounts. A methyl ether corresponding to HSQ was detected when methanol was present in the incubation buffer. These compounds are the expected "solvolysis" products from PSPP. They provide strong support for mechanisms that propose cyclopropylcarbinyl cations as intermediates in the SQase-catalyzed rearrangement of PSPP to SQ and unambiguously demonstrate that the catalytic machinery of SQase is capable of synthesizing a variety of irregular isoprenoids.     

Synthesis and evaluation of 1-deoxy-D-xylulose 5-phosphoric acid analogues as alternate substrates for methylerythritol phosphate synthase

[structure: see text] Four deoxyxylulose phosphate (DXP) analogues were synthesized and evaluated as substrates/inhibitors for methylerythritol phosphate (MEP) synthase. In analogues CF(3)-DXP (1), CF(2)-DXP (2), and CF-DXP (3), the three methyl hydrogens at C1 of DXP were sequentially replaced by fluorine. In the fourth analogue, Et-DXP (4), the methyl group in DXP was replaced by an ethyl moiety. Analogues 1, 2, and 4 were not substrates for MEP synthase under normal catalytic conditions and were instead modest inhibitors with IC(50) values of 2.0, 3.4, and 6.2 mM, respectively. In contrast, 3 was a good substrate (k(cat) = 38 s(-)(1), K(m) = 227 muM) with a turnover rate similar to that of the natural substrate. These results are consistent with a retro-aldol/aldol mechanism rather than an alpha-ketol rearrangement for the enzyme-catalyzed conversion of DXP to MEP.     

Large-amplitude plasma wave generation with a high-intensity short-pulse beat wave

A short-pulse laser beat wave scheme for advanced particle accelerator applications is examined. A short, intense (3-ps, >10(18)-W cm(-2)) two-frequency laser pulse is produced by use of a modified chirped-pulse amplification scheme and is shown to produce relativistic plasma waves during interactions with low-density plasmas. The generation of plasma waves was observed by measurement of forward Raman scattering. Resonance was found to occur at an electron density many times that expected, owing to ponderomotive displacement of plasma within the focal region.