Isopentenyl diphosphate:dimethylallyl diphosphate (IPP:DMAPP) isomerase catalyses a crucial activation step in the isoprenoid biosynthesis pathway. This enzyme is responsible for the isomerization of the carbon±carbon double bond of IPP to create the potent electrophile DMAPP. DMAPP then alkylates other molecules, including IPP, to initiate the extraordinary variety of isoprenoid compounds found in nature. The crystal structures of free and metal-bound Escherichia coli IPP isomerase reveal critical active site features underlying its catalytic mechanism. The enzyme requires one Mn 2+ or Mg 2+ ion to fold in its active conformation, forming a distorted octahedral metal coordination site composed of three histidines and two glutamates and located in the active site. Two critical residues, C67 and E116, face each other within the active site, close to the metal-binding site. The structures are compatible with a mechanism in which the cysteine initiates the reaction by protonating the carbon±carbon double bond, with the antarafacial rearrangement ultimately achieved by one of the glutamates involved in the metal coordination sphere. W161 may stabilize the highly reactive carbocation generated during the reaction through quadrupole± charge interaction.

Xylanases are hemicellulases that hydrolyze the internal beta-1,4-glycoside bonds of xylan. The extracellular thermostable endo-1,4-beta-xylanase (EC 3.2.1.8; XT6) produced by the thermophilic bacterium Geobacillus stearothermophilus T-6 was shown to bleach pulp optimally at pH 9 and 338 K and was successfully used in a large-scale biobleaching mill trial. The xylanase gene was cloned and sequenced. The mature enzyme consists of 379 amino acids, with a calculated molecular weight of 43 808 Da and a pI of 9.0. Crystallographic studies of XT6 were performed in order to study the mechanism of catalysis and to provide a structural basis for the rational introduction of enhanced thermostability by site-specific mutagenesis. XT6 was crystallized in the primitive trigonal space group P3(2)21, with unit-cell parameters a = b = 112.9, c = 122.7 A. A full diffraction data set for wild-type XT6 has been measured to 2.4 A resolution on flash-frozen crystals using synchrotron radiation. A fully exchanged selenomethionyl XT6 derivative (containing eight Se atoms per XT6 molecule) was also prepared and crystallized in an isomorphous crystal form, providing full selenium MAD data at three wavelengths and enabling phase solution and structure determination. The structure of wild-type XT6 was refined at 2.4 A resolution to a final R factor of 15.6% and an R(free) of 18.6%. The structure demonstrates that XT6 is made up of an eightfold TIM-barrel containing a deep active-site groove, consistent with its 'endo' mode of action. The two essential catalytic carboxylic residues (Glu159 and Glu265) are located at the active site within 5.5 A of each other, as expected for 'retaining' glycoside hydrolases. A unique subdomain was identified in the carboxy-terminal part of the enzyme and was suggested to have a role in xylan binding. The three-dimensional structure of XT6 is of great interest since it provides a favourable starting point for the rational improvement of its already high thermal and pH stabilities, which are required for a number of biotechnological and industrial applications.

The structure of cytochrome f includes an internal chain of five water molecules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range of photosynthetic organisms from higher plants, algae, and cyanobacteria. The in vivo electron transfer capability of Chlamydomonas reinhardtii cytochrome f was impaired in site-directed mutants of the conserved Asn and Gln residues that form hydrogen bonds with water molecules of the internal chain [Ponamarev, M. V., and Cramer, W. A. (1998) Biochemistry 37, 17199-17208]. The 251-residue extrinsic functional domain of C. reinhardtii cytochrome f was expressed in Escherichia coli without the 35 C-terminal residues of the intact cytochrome that contain the membrane anchor. Crystal structures were determined for the wild type and three "water chain" mutants (N168F, Q158L, and N153Q) having impaired photosynthetic and electron transfer function. The mutant cytochromes were produced, folded, and assembled heme at levels identical to that of the wild type in the E. coli expression system. N168F, which had a non-photosynthetic phenotype and was thus most affected by mutational substitution, also had the greatest structural perturbation with two water molecules (W4 and W5) displaced from the internal chain. Q158L, the photosynthetic mutant with the largest impairment of in vivo electron transfer, had a more weakly bound water at one position (W1). N153Q, a less impaired photosynthetic mutant, had an internal water chain with positions and hydrogen bonds identical to those of the wild type. The structure data imply that the waters of the internal chain, in addition to the surrounding protein, have a significant role in cytochrome f function.

FdVI from Rhodobacter capsulatus is structurally related to a group of [2Fe-2S] ferredoxins involved in iron-sulfur cluster biosynthesis. Comparative genomics suggested that FdVI and orthologs found in alpha-Proteobacteria are involved in this process. Here, the crystal structure of FdVI has been determined for both the oxidized and the reduced protein. The [2Fe-2S] cluster lies 6 A below the protein surface in a hydrophobic pocket without access to the solvent. This particular cluster environment might explain why the FdVI midpoint redox potential (-306 mV at pH 8.0) did not show temperature or ionic strength dependence. Besides the four cysteines that bind the cluster, FdVI features an extra cysteine which is located close to the S1 atom of the cluster and is oriented in a position such that its thiol group points towards the solvent. Upon reduction, the general fold of the polypeptide chain was almost unchanged. The [2Fe-2S] cluster underwent a conformational change from a planar to a distorted lozenge. In the vicinity of the cluster, the side chain of Met24 was rotated by 180 degrees , bringing its S atom within hydrogen-bonding distance of the S2 atom of the cluster. The reduced molecule also featured a higher content of bound water molecules, and more extensive hydrogen-bonding networks compared with the oxidized molecule. The unique conformational changes observed in FdVI upon reduction are discussed in the light of structural studies performed on related ferredoxins.

The pseudo-reverse reaction of Pseudomonas aeruginosa catabolic ornithine carbamoyltransferase in which arsenate is first coupled to citrulline followed by elimination of carbamylarsenate has been studied. Arsenate and citrulline saturation curves are sigmoidal. The different responsiveness of the transcarbamoylase to isosteric and allosteric ligands was examined both in the forward reaction, the carbamoylation of ornithine, and in the pseudo-reverse reaction, the arsenolytic cleavage of citrulline. Nucleoside monophosphates and polyamines that act as allosteric activators and inhibitors, respectively, on the carbamoylation reaction have the same effect on the rate of the arsenolytic cleavage of citrulline. ATP and other nucleoside triphosphates were found to stimulate enzyme activity at low carbamoylphosphate concentration with little influence on the carbamoylphosphate concentration at half-maximum velocity as well as on the cooperative index. When measuring the initial rate of the reverse reaction, the arsenolytic cleavage of citrulline, ATP was found to be a weak inhibitor, whereas CTP still stimulates the reaction and UTP was without influence. This unidirectional inhibition or activation phenomenon is likely apparent since initial studies were conducted and no consideration was given to equilibrium conditions. Regulation of catabolic OTCase by nucleoside triphosphates is without physiological meaning. In contrast, stimulation by nucleoside monophosphates may indicate that energy limitation could promote the synthesis and activity of the catabolic enzyme.