Comparison of multiple gene assembly methods for metabolic engineering

A universal, rapid DNA assembly method for efficient multigene plasmid construction is important for biological research and for optimizing gene expression in industrial microbes. Three different approaches to achieve this goal were evaluated. These included creating long complementary extensions using a uracil-DNA glycosylase technique, overlap extension polymerase chain reaction, and a SfiI-based ligation method. SfiI ligation was the only successful approach for assembling large DNA fragments that contained repeated homologous regions. In addition, the SfiI method has been improved over a similar, previous published technique so that it is more flexible and does not require polymerase chain reaction to incorporate adaptors. In the present study, Saccharomyces cerevisiae genes TAL1, TKL1, and PYK1 under control of the 6-phosphogluconate dehydrogenase promoter were successfully ligated together using multiple unique SfiI restriction sites. The desired construct was obtained 65% of the time during vector construction using four-piece ligations. The SfiI method consists of three steps: first a SfiI linker vector is constructed, whose multiple cloning site is flanked by two three-base linkers matching the neighboring SfiI linkers on SfiI digestion; second, the linkers are attached to the desired genes by cloning them into SfiI linker vectors; third, the genes flanked by the three-base linkers, are released by SfiI digestion. In the final step, genes of interest are joined together in a simple one-step ligation.     

The diverse roles of flavin coenzymes--nature's most versatile thespians

Flavin coenzymes play a variety of roles in biological systems. This Perspective highlights the chemical versatility of flavins by reviewing research on five flavoenzymes that have been studied in our laboratory. Each of the enzymes discussed in this review [the acyl-CoA dehydrogenases (ACDs), CDP-6-deoxy-l-threo-d-glycero-4-hexulose-3-dehydrase reductase (E3), CDP-4-aceto-3,6-dideoxygalactose synthase (YerE), UDP-galactopyranose mutase (UGM), and type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2)] utilizes flavin in a distinct role. In particular, the catalytic mechanisms of two of these enzymes, UGM and IDI-2, may involve novel flavin chemistry.     

Analysis of UDP-d-Apiose/UDP-d-Xylose Synthase-Catalyzed Conversion of UDP-d-Apiose Phosphonate to UDP-d-Xylose Phosphonate: Implications for a Retroaldol-Aldol Mechanism

UDP-d-apiose/UDP-d-xylose synthase (AXS) catalyzes the conversion of UDP-d-glucuronic acid to UDP-d-apiose and UDP-d-xylose. An acetyl-protected phosphonate analogue of UDP-d-apiose was synthesized and used in an in situ HPLC assay to demonstrate for the first time the ability of AXS to interconvert the two reaction products. Density functional theory calculations provided insight into the energetics of this process and the apparent inability of AXS to catalyze the conversion of UDP-d-xylose to UDP-d-apiose. The data suggest that this observation is unlikely to be due to an unfavorable equilibrium but rather results from substrate inhibition by the most stable chair conformation of UDP-d-xylose. The detection of xylose cyclic phosphonate as the turnover product reveals significant new details about the AXS-catalyzed reaction and supports the proposed retroaldol-aldol mechanism of catalysis.     

Mechanistic studies of the radical S-adenosyl-L-methionine enzyme DesII: EPR characterization of a radical intermediate generated during its catalyzed dehydrogenation of TDP-D-quinovose

DesII, a radical S-adenosyl-l-methionine (SAM) enzyme from Streptomyces venezuelae, catalyzes the deamination of TDP-4-amino-4,6-dideoxy-D-glucose to TDP-3-keto-4,6-dideoxy-D-glucose in the desosamine biosynthetic pathway. DesII can also catalyze the dehydrogenation of TDP-D-quinovose to the corresponding 3-keto sugar. Similar to other radical SAM enzymes, DesII catalysis has been proposed to proceed via a radical mechanism. This hypothesis is now confirmed by EPR spectroscopy with the detection of a TDP-D-quinovose radical intermediate having a g-value of 2.0025 with hyperfine coupling to two spin 1/2 nuclei, each with a splitting constant of 33.6 G. A significant decrease in the EPR line width is observed when the radical is generated in reactions conducted in D(2)O versus H(2)O. These results are consistent with a C3 α-hydroxyalkyl radical in which the p-orbital harboring the unpaired electron spin at C3 is periplanar with the C-H bonds at both C2 and C4.     

Pulsed electron paramagnetic resonance experiments identify the paramagnetic intermediates in the pyruvate ferredoxin oxidoreductase catalytic cycle

Pyruvate ferredoxin oxidoreductase (PFOR) is central to the anaerobic metabolism of many bacteria and amitochondriate eukaryotes. PFOR contains thiamine pyrophosphate (TPP) and three [4Fe-4S] clusters, which link pyruvate oxidation to reduction of ferredoxin. In the PFOR reaction, TPP reacts with pyruvate to form lactyl-TPP, which undergoes decarboxylation to form a hydroxyethyl-TPP (HE-TPP) intermediate. One electron is then transferred from HE-TPP to one of the three [4Fe-4S] clusters to form an HE-TPP radical and a [4Fe-4S]1+ intermediate. Pulsed EPR methods have been used to measure the distance between the HE-TPP radical and the [4Fe-4S]1+ cluster to which it is coupled. Computational analysis including the PFOR crystal structure and the spin distribution in the HE-TPP radical and in the reduced [4Fe-4S] cluster demonstrates that the distance between the HE-TPP radical and the medial cluster B matches the experimentally determined dipolar interaction, while one of the other two clusters is too close and the other is too far away. These results clearly demonstrate that it is the medial cluster (cluster B) that is reduced. Thus, rapid electron transfer occurs through the electron-transfer chain, which leaves an oxidized proximal cluster poised to accept an electron from the HE-TPP radical in the subsequent reaction step.