A number of X-ray analyses of an enzyme involved in a key early stage of tetrapyrrole biosynthesis are reported. Two structures of human 5-aminolaevulinate dehydratase (ALAD), native and recombinant, have been determined at 2.8 Å resolution, showing that the enzyme adopts an octameric quaternary structure in accord with previously published analyses of the enzyme from a range of other species. However, this is in contrast to the finding that a disease-related F12L mutant of the human enzyme uniquely forms hexamers [Breinig et al. (2003), Nature Struct. Biol. 10, 757-763]. Monomers of all ALADs adopt the TIM-barrel fold; the subunit conformation that assembles into the octamer includes the N-terminal tail of one monomer curled around the (α/β)8 barrel of a neighbouring monomer. Both crystal forms of the human enzyme possess two monomers per asymmetric unit, termed A and B. In the native enzyme there are a number of distinct structural differences between the A and B monomers, with the latter exhibiting greater disorder in a number of loop regions and in the active site. In contrast, the second monomer of the recombinant enzyme appears to be better defined and the active site of both monomers clearly possesses a zinc ion which is bound by three conserved cysteine residues. In native human ALAD, the A monomer also has a ligand resembling the substrate ALA which is covalently bound by a Schiff base to one of the active-site lysines (Lys252) and is held in place by an ordered active-site loop. In contrast, these features of the active-site structure are disordered or absent in the B subunit of the native human enzyme. The octameric structure of the zinc-dependent ALAD from the hyperthermophile Pyrobaculum calidifontis is also reported at a somewhat lower resolution of 3.5 Å. Finally, the details are presented of a high-resolution structure of the Escherichia coli ALAD enzyme co-crystallized with a noncovalently bound moiety of the product, porphobilinogen (PBG). This structure reveals that the pyrrole side-chain amino group is datively bound to the active-site zinc ion and that the PBG carboxylates interact with the enzyme via hydrogen bonds and salt bridges with invariant residues. A number of hydrogen-bond interactions that were previously observed in the structure of yeast ALAD with a cyclic intermediate resembling the product PBG appear to be weaker in the new structure, suggesting that these interactions are only optimal in the transition state.

The diffraction images which allowed the original 2.1 Angstrom resolution structure determination of Escherichia coli ALAD co-crystallised with a non-covalently bound moiety of the product, porphobilinogen (PBG), are presented.

Introduction Norovirus is the most common cause of viral gastroenteritis in man. It affects approximately 267 million people/annum and, although usually self-limiting, infection is still associated with around 200,000 deaths/annum. Norovirus infection has a significant, detrimental impact on societal infrastructure; it is the leading pathogen responsible for forced-ward closures in the NHS. There are no specific treatments available; the most promising target for antiviral therapy is noroviral 3 C protease (3 CLpro) which processes the polyprotein essential for the production of viral proteins. Inhibiting 3 CLpro would stop viral replication. This study focused on the function of 3 CLpro, especially the cleavage sites from its precursor. Methods The wild-type (WT) 3 CLpro sequence was altered with a cysteine to alanine base substitution to create a 3 CLpro mutant with greatly reduced catalytic activity. The mutant was expressed in E.coli and purified using ion exchange and size-filtration chromatography. Protein expression was confirmed by gel electrophoresis and Western blotting. The specificity of 3 CLpro was studied using a spectrophotometric assay and the rates of reaction of mutant and WT 3 CLpro with substrate were compared. The chemical composition of mutant and WT 3 CLpro were examined using mass spectroscopy. Results Western blot analysis showed multiple bands indicating that both WT and mutant 3 CLpro appeared to be cleaved out of their precursor. Enzyme kinetic studies, however, confirmed that mutant 3 CLpro had negligible catalytic activity; the WT 3 CLpro’s turnover rate of catalytic activity was 25 times faster than that of mutant 3 CLpro. Mass spectrometry of mutant 3 CLpro generated a mass spectrum and transformed to a protein mass of 18,747.5. This confirmed the identity of noroviral 3 CLpro, which is 19 kDA. Conclusion Mutant 3 CLpro was still cleaved out of its precursor despite the fact that it has negligible catalytic activity. The most likely explanation is that the cleavage was effected by an E.coli protease, possibly a metalloprotease, acting at the upstream and downstream boundaries thereby releasing the processed mutant 3 CLpro. The fact that mutant 3 CLpro can be cleaved out of its precursor by host-cell proteases raises the possibility that WT 3 CLpro could also be processed by exogenous proteases, rather than cleaving itself from its precursor. If this action by host cell proteases occurs in vivo, then it might indicate that the norovirus 3 CLpro is only needed for the cleavage of polyproteins within the newly formed virions, which do not have access to host cell proteases. This has significant implications for future research. Disclosure of Interest None Declared

X-ray diffraction images for Escherichia coli 5-aminolevulinic acid dehydratase (ALAD) which was crystallised in the presence of the inhibitor levulinic acid (15 mM) and bismuth nitrate (1 mM). The data were collected at beamline 9.6 at the SRS Daresbury Laboratory (UK) on 10th March 1994 using a 30 cm Marresearch image plate detector, a crystal temperature of 100 K, a wavelength of 0.88 Å and a crystal-to-detector distance was 300 mm. The oscillation angle was 2.5 degrees and 21 images were collected at constant dose in the time available. A wax image for determining the direct beam position was taken with the detector at a distance of 400 mm.

The protein calexcitin was originally identified in molluscan photoreceptor neurons as a 20 kDa molecule which was up-regulated and phosphorylated following a Pavlovian conditioning protocol. Subsequent studies showed that calexcitin regulates the voltage-dependent potassium channel and the calcium-dependent potassium channel as well as causing the release of calcium ions from the endoplasmic reticulum (ER) by binding to the ryanodine receptor. A crystal structure of calexcitin from the squid Loligo pealei showed that the fold is similar to that of another signalling protein, calmodulin, the N- and C-terminal domains of which are known to separate upon calcium binding, allowing interactions with the target protein. Phosphorylation of calexcitin causes it to translocate to the cell membrane, where its effects on membrane excitability are exerted and, accordingly, L. pealei calexcitin contains two protein kinase C phosphorylation sites (Thr61 and Thr188). Thr-to-Asp mutations which mimic phosphorylation of the protein were introduced and crystal structures of the corresponding single and double mutants were determined, which suggest that the C-terminal phosphorylation site (Thr188) exerts the greatest effects on the protein structure. Extensive NMR studies were also conducted, which demonstrate that the wild-type protein predominantly adopts a more open conformation in solution than the crystallographic studies have indicated and, accordingly, normal-mode dynamic simulations suggest that it has considerably greater capacity for flexible motion than the X-ray studies had suggested. Like calmodulin, calexcitin consists of four EF-hand motifs, although only the first three EF-hands of calexcitin are involved in binding calcium ions; the C-terminal EF-hand lacks the appropriate amino acids. Hence, calexcitin possesses two functional EF-hands in close proximity in its N-terminal domain and one functional calcium site in its C-terminal domain. There is evidence that the protein has two markedly different affinities for calcium ions, the weaker of which is most likely to be associated with binding of calcium ions to the protein during neuronal excitation. In the current study, site-directed mutagenesis has been used to abolish each of the three calcium-binding sites of calexcitin, and these experiments suggest that it is the single calcium-binding site in the C-terminal domain of the protein which is likely to have a sensory role in the neuron.

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid. This enzyme is a very unusual dioxygenase in that it cleaves a C-C bond in a substituent of the aromatic ring rather than within the ring itself. Whilst it has been shown that DAD is a tetramer in solution, the recently solved crystal structure of the Alcaligenes sp. 4HAP enzyme was in fact dimeric rather than tetrameric. Since the use of limited chymotrypsinolysis, which apparently results in removal of the first 20 or so N-terminal residues of DAD, was necessary for crystallization of the protein, it was investigated whether this was responsible for the change in its oligomerization state. Gel-filtration and analytical ultracentrifugation studies were conducted, which confirmed that chymotrypsinolysed DAD has an apparent molecular weight of around 40 kDa, corresponding to a dimer. In contrast, the native enzyme has a molecular weight in the 70-80 kDa region, as expected for the tetramer. The structural basis for tetramerization has been investigated by the use of several docking servers, and the results are remarkably consistent with the tetrameric structure of a homologous cupin protein from Ralstonia eutropha (PDB entry 3ebr).

The enzyme 2,4'-dihydroxyacetophenone dioxygenase (or DAD) catalyses the conversion of 2,4'-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C-C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits each containing nonhaem iron and its sequence suggests that it belongs to the cupin family of dioxygenases. By the use of limited chymotrypsinolysis, the DAD enzyme from Alcaligenes sp. 4HAP has been crystallized in a form that diffracts synchrotron radiation to a resolution of 2.2 Å.

The enzyme 2,4′-dihydroxyacetophenone dioxygenase (DAD) catalyses the conversion of 2,4′-dihydroxyacetophenone to 4-hydroxybenzoic acid and formic acid with the incorporation of molecular oxygen. Whilst the vast majority of dioxygenases cleave within the aromatic ring of the substrate, DAD is very unusual in that it is involved in C—C bond cleavage in a substituent of the aromatic ring. There is evidence that the enzyme is a homotetramer of 20.3 kDa subunits, each containing nonhaem iron, and its sequence suggests that it belongs to the cupin family of dioxygenases. In this paper, the first X-ray structure of a DAD enzyme from the Gram-negative bacterium Alcaligenes sp. 4HAP is reported, at a resolution of 2.2 Å. The structure establishes that the enzyme adopts a cupin fold, forming dimers with a pronounced hydrophobic interface between the monomers. The catalytic iron is coordinated by three histidine residues (76, 78 and 114) within a buried active-site cavity. The iron also appears to be tightly coordinated by an additional ligand which was putatively assigned as a carbonate dianion since this fits the electron density optimally, although it might also be the product formate. The modelled carbonate is located in a position which is highly likely to be occupied by the α-hydroxyketone group of the bound substrate during catalysis. Modelling of a substrate molecule in this position indicates that it will interact with many conserved amino acids in the predominantly hydrophobic active-site pocket where it undergoes peroxide radical-mediated heterolysis.