The structure of a prokaryotic feruloyl-CoA hydratase-lyase from a lignin-degrading consortium with high oligomerization stability under extreme pHs.

In the context of increasing demand for renewable alternatives of fuels and chemicals, the valorization of lignin emerges as a value-adding strategy in biorefineries and an alternative to petroleum-derived molecules. One of the compounds derived from lignin is ferulic acid (FA), which can be converted into valuable molecules such as vanillin. In microorganisms, FA biotransformation into vanillin can occur via a two-step reaction catalyzed by the sequential activity of a feruloyl-CoA synthetase (FCS) and an feruloyl-CoA hydratase-lyase (FCHL), which could be exploited industrially. In this study, a prokaryotic FCHL derived from a lignin-degrading microbial consortium (named LM-FCHL) was cloned, successfully expressed in soluble form and purified. The crystal structure was solved and refined at 2.1 Å resolution. The LM-FCHL is a hexamer composed of a dimer of trimers, which showed to be quite stable under extreme pH conditions. Finally, small angle X-ray scattering corroborates the hexameric state in solution and indicates flexibility in the protein structure. The present study contributes to the field of lignin valorization to valuable molecules by establishing the biophysical and structural characterization for a novel FCHL member of unique characteristics.

Fused dimerization increases expression, solubility, and activity of bacterial dehydratase enzymes.

FabA and FabZ are the two dehydratase enzymes in Escherichia coli that catalyze the dehydration of acyl intermediates in the biosynthesis of fatty acids. Both enzymes form obligate dimers in which the active site contains key amino acids from both subunits. While FabA is a soluble protein that has been relatively straightforward to express and to purify from cultured E. coli, FabZ has shown to be mostly insoluble and only partially active. In an effort to increase the solubility and activity of both dehydratases, we made constructs consisting of two identical subunits of FabA or FabZ fused with a naturally occurring peptide linker, so as to force their dimerization. The fused dimer of FabZ (FabZ-FabZ) was expressed as a soluble enzyme with an ninefold higher activity in vitro than the unfused FabZ. This construct exemplifies a strategy for the improvement of enzymes from the fatty acid biosynthesis pathways, many of which function as dimers, catalyzing critical steps for the production of fatty acids.

Yeast two-hybrid liquid screening.

Yeast two-hybrid (YTH) method consists of a genetic trap that selects for "prey" cDNA products within a library that interact with a "bait" protein of interest. Here, we provide a protocol for YTH screening using a liquid medium screening method, which improves the sensitivity of this technique and streamlines the laborious classic screening in solid medium plates. The method uses a simple series of dilutions with established yeast strains transformed with diverse baits and complex cDNA libraries. This allows for prompt detection of positive clones revealed by liquid growth, due to activation of HIS3 reporter gene. Activation of a second reporter gene and reconstruction of the YTH interaction is highly reproducible using this system. This approach can either be performed using culture flasks or deep-well 96-well plates and the number of interactions obtained is similar, when compared to the classic method. In addition, the liquid screening method is faster and more economical for YTH screening and has the added benefit of automation if 96-well plates are used.

Identification of novel protein domains required for the expression of an active dehydratase fragment from a polyunsaturated fatty acid synthase.

Polyunsaturated fatty acids (PUFAs) are made in some strains of deep-sea bacteria by multidomain proteins that catalyze condensation, ketoreduction, dehydration, and enoyl-reduction. In this work, we have used the Udwary-Merski Algorithm sequence analysis tool to define the boundaries that enclose the dehydratase (DH) domains in a PUFA multienzyme. Sequence analysis revealed the presence of four areas of high structure in a region that was previously thought to contain only two DH domains as defined by FabA-homology. The expression of the protein fragment containing all four protein domains resulted in an active enzyme, while shorter protein fragments were not soluble. The tetradomain fragment was capable of catalyzing the conversion of crotonyl-CoA to ß-hydroxybutyryl-CoA efficiently, as shown by UV absorbance change as well as by chromatographic retention of reaction products. Sequence alignments showed that the two novel domains contain as much sequence conservation as the FabA-homology domains, suggesting that they too may play a functional role in the overall reaction. Structure predictions revealed that all domains belong to the hotdog protein family: two of them contain the active site His70 residue present in FabA-like DHs, while the remaining two do not. Replacing the active site His residues in both FabA domains for Ala abolished the activity of the tetradomain fragment, indicating that the DH activity is contained within the FabA-homology regions. Taken together, these results provide a first glimpse into a rare arrangement of DH domains which constitute a defining feature of the PUFA synthases.

Tellurite-mediated disabling of [4Fe-4S] clusters of Escherichia coli dehydratases.

The tellurium oxyanion tellurite is toxic for most organisms and it seems to alter a number of intracellular targets. In this work the toxic effects of tellurite upon Escherichia coli [4Fe-4S] cluster-containing dehydratases was studied. Reactive oxygen species (ROS)-sensitive fumarase A (FumA) and aconitase B (AcnB) as well as ROS-resistant fumarase C (FumC) and aconitase A (AcnA) were assayed in cell-free extracts from tellurite-exposed cells in both the presence and absence of oxygen. While over 90 % of FumA and AcnB activities were lost in the presence of oxygen, no enzyme inactivation was observed in anaerobiosis. This result was not dependent upon protein biosynthesis, as determined using translation-arrested cells. Enzyme activity of purified FumA and AcnB was inhibited when exposed to an in vitro superoxide-generating, tellurite-reducing system (ITRS). No inhibitory effect was observed when tellurite was omitted from the ITRS. In vivo and in vitro reconstitution experiments with tellurite-damaged FumA and AcnB suggested that tellurite effects involve [Fe-S] cluster disabling. In fact, after exposing FumA to ITRS, released ferrous ion from the enzyme was demonstrated by spectroscopic analysis using the specific Fe(2+) chelator 2,2'-bipyridyl. Subsequent spectroscopic paramagnetic resonance analysis of FumA exposed to ITRS showed the characteristic signal of an oxidatively inactivated [3Fe-4S](+) cluster. These results suggest that tellurite inactivates enzymes of this kind via a superoxide-dependent disabling of their [4Fe-4S] catalytic clusters.

Cloning and sequence analysis of the Candida utilis HIS3 gene.

A DNA fragment, carrying the Candida utilis HIS3 gene, has been isolated from a genomic DNA library by complementation of the E. coli hisB mutant. Its nucleotide sequence was determined and it predicts a single open reading frame of 675 bp (224 aa). The deduced amino acid sequence is highly homologous to other yeast and fungi HIS3 genes.

High resolution X-ray structure of dTDP-glucose 4,6-dehydratase from Streptomyces venezuelae.

Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found in some macrolide antibiotics. In Streptomyces venezuelae, there are seven genes required for the biosynthesis of this unusual sugar. One of the genes, desIV, codes for a dTDP-glucose 4,6-dehydratase, which is referred to as DesIV. The reaction mechanisms for these types of dehydratases are quite complicated with proton abstraction from the sugar 4'-hydroxyl group and hydride transfer to NAD+, proton abstraction at C-5, and elimination of the hydroxyl group at C-6 of the sugar, and finally return of a proton to C-5 and a hydride from NADH to C-6. Here we describe the cloning, overexpression, and purification, and high resolution x-ray crystallographic analysis to 1.44 A of wild-type DesIV complexed with dTDP. Additionally, for this study, a double site-directed mutant protein (D128N/E129Q) was prepared, crystallized as a complex with NAD+ and the substrate dTDP-glucose and its structure determined to 1.35 A resolution. In DesIV, the phenolate group of Tyr(151) and O(gamma) of Thr(127) lie at 2.7 and 2.6 A, respectively from the 4'-hydroxyl group of the dTDP-glucose substrate. The side chain of Asp(128) is in the correct position to function as a general acid for proton donation to the 6'-hydroxyl group while the side chain of Glu(129) is ideally situated to serve as the general base for proton abstraction at C-5. This investigation provides further detailed information for understanding the exquisite chemistry that occurs in these remarkable enzymes.

Comparison of different crystal forms of 3-dehydroquinase from Salmonella typhi and its implication for the enzyme activity.

The type I 3-dehydroquinate dehydratase (DHQase) which catalyses the reversible dehydration of 3-dehydroquinic acid to 3-dehydroshikimic acid is involved in the shikimate pathway for the biosynthesis of aromatic compounds. The shikimate pathway is absent in mammals, which makes structural information about DHQase vital for the rational design of antimicrobial drugs and herbicides. The crystallographic structure of the type I DHQase from Salmonella typhi has now been determined for the native form at 1.78 A resolution (R = 19.9%; R(free) = 24.7%). The structure of the modified enzyme to which the product has been covalently bound has also been determined but in a different crystal form (2.1 A resolution; R = 17.7%; R(free) = 24.5%). An analysis of the three available crystal forms has provided information about the physiological dimer interface. The enzyme relies upon the closure of a lid-like loop to complete its active site. As the lid-loop tends to stay in the closed position, dimerization appears to play a role in biasing the arrangement of the loop towards its open position, thus facilitating substrate access.