Oxidation of lysozyme by iodine: identification and properties of an oxindolyl ester intermediate: evidence for participation of glutamic acid 35 in catalysis

The first product formed in the iodine oxidation of tryptophan 108 of lysozyme has a transition temperature more than 20 deg. C higher than that of native and oxindolealanine 108-lysozyme. Irreversible rapid conversion to the oxindole follows unfolding. The spectrum of the oxidized residue of the intermediate resembles that of tryptophan. The iodine oxidation of tryptophan 108 is faster than that of N-acetyltryptophan ethyl ester. These and other aspects of the lysozyme-iodine reaction are explained by the formation, possibly concerted with oxidation, of the oxindolyl ester of glutamic acid 35. The data accord with results of high-resolution crystallographic analysis (Beddell &; Blake, 1970). Ester 108-lysozyme binds substrate like the native enzyme but retains less than 0.1% of the native activity. These results and the crystallographic data demonstrate catalytic function for glutamic acid 35. Oxindolealanine 108-lysozyme binds substrate only weakly. Introduction of an ester crosslink adds more than 6 kcal to the stability of lysozyme.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly 2H, 13C, 15N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

Solution NMR of signal peptidase, a membrane protein

Useful solution nuclear magnetic resonance (NMR) data can be obtained from full-length, enzymatically active type I signal peptidase (SPase I), an integral membrane protein, in detergent micelles. Signal peptidase has two transmembrane segments, a short cytoplasmic loop, and a 27-kD C-terminal catalytic domain. It is a critical component of protein transport systems, recognizing and cleaving amino-terminal signal peptides from preproteins during the final stage of their export. Its structure and interactions with the substrate are of considerable interest, but no three-dimensional structure of the whole protein has been reported. The structural analysis of intact membrane proteins has been challenging and only recently has significant progress been achieved using NMR to determine membrane protein structure. Here we employ NMR spectroscopy to study the structure of the full-length SPase I in dodecylphosphocholine detergent micelles. HSQC-TROSY spectra showed resonances corresponding to approximately 3/4 of the 324 residues in the protein. Some sequential assignments were obtained from the 3D HNCACB, 3D HNCA, and 3D HN(CO) TROSY spectra of uniformly ²H, ¹³C, ¹⁵N-labeled full-length SPase I. The assigned residues suggest that the observed spectrum is dominated by resonances arising from extramembraneous portions of the protein and that the transmembrane domain is largely absent from the spectra. Our work elucidates some of the challenges of solution NMR of large membrane proteins in detergent micelles as well as the future promise of these kinds of studies.     

Roles of active site residues in Pseudomonas aeruginosa phosphomannomutase/phosphoglucomutase

In Pseudomonas aeruginosa, the dual-specificity enzyme phosphomannomutase/phosphoglucomutase catalyzes the transfer of a phosphoryl group from serine 108 to the hydroxyl group at the 1-position of the substrate, either mannose 6-P or glucose 6-P. The enzyme must then catalyze transfer of the phosphoryl group on the 6-position of the substrate back to the enzyme. Each phosphoryl transfer is expected to require general acid-base catalysis, provided by amino acid residues at the enzyme active site. An extensive survey of the active site residues by site-directed mutagenesis failed to identify a single key residue that mediates the proton transfers. Mutagenesis of active site residues Arg20, Lys118, Arg247, His308, and His329 to residues that do not contain ionizable groups produced proteins for which V(max) was reduced to 4-12% of that of the wild type. The fact that no single residue decreased catalytic activity more significantly, and that several residues had similar effects on V(max), suggested that the ensemble of active site amino acids act by creating positive electrostatic potential, which serves to depress the pK of the substrate hydroxyl group so that it binds in ionized form at the active site. In this way, the necessity of positioning the reactive hydroxyl group near a specific amino acid residue is avoided, which may explain how the enzyme is able to promote catalysis of both phosphoryl transfers, even though the 1- and 6-positions do not occupy precisely the same position when the substrate binds in the two different orientations in the active site. When Ser108 is mutated, the enzyme retains a surprising amount of activity, which has led to the suggestion that an alternative residue becomes phosphorylated in the absence of Ser108. (31)P NMR spectra of the S108A protein confirm that it is phosphorylated. Although the S108A/H329N protein had no detectable catalytic activity, the (31)P NMR spectra were not consistent with a phosphohistidine residue.     

The active site and substrates binding mode of malonyl-CoA synthetase determined by transferred nuclear Overhauser effect spectroscopy, site-directed mutagenesis, and comparative modeling studies

The active sites and substrate bindings of Rhizobium trifolii molonyl-CoA synthetase (MCS) catalyzing the malonyl-CoA formation from malonate and CoA have been determined based on NMR spectroscopy, site-directed mutagenesis, and comparative modeling methods. The MCS-bound conformation of malonyl-CoA was determined from two-dimensional-transferred nuclear Overhauser effect spectroscopy data. MCS protein folds into two structural domains and consists of 16 alpha-helices, 24 beta-strands, and several long loops. The core active site was determined as a wide cleft close to the end of the small C-terminal domain. The catalytic substrate malonate is placed between ATP and His206 in the MCS enzyme, supporting His206 in its catalytic role as it generates reaction intermediate, malonyl-AMP. These findings are strongly supported by previous biochemical data, as well as by the site-directed mutagenesis data reported here. This structure reveals the biochemical role as well as the substrate specificity that conservative residues of adenylate-forming enzymes have.     

Phosphorylation in the Catalytic Cleft Stabilizes and Attracts Domains of a Phosphohexomutase

Phosphorylation can modulate the activities of enzymes. The phosphoryl donor in the catalytic cleft of α-D-phosphohexomutases is transiently dephosphorylated while the reaction intermediate completes a 180° reorientation within the cleft. The phosphorylated form of 52 kDa bacterial phosphomannomutase/phosphoglucomutase is less accessible to dye or protease, more stable to chemical denaturation, and widely stabilized against NMR-detected hydrogen exchange across the core of domain 3 to juxtaposed domain 4 (each by ≥1.3 kcal/mol) and parts of domains 1 and 2. However, phosphorylation accelerates hydrogen exchange in specific regions of domains 1 and 2, including a metal-binding residue in the active site. Electrostatic field lines reveal attraction across the catalytic cleft between phosphorylated Ser-108 and domain 4, but repulsion when Ser-108 is dephosphorylated. Molecular dynamics (MD) simulated the dephosphorylated form to be expanded due to enhanced rotational freedom of domain 4. The contacts and fluctuations of the MD trajectories enabled correct simulation of more than 80% of sites that undergo either protection or deprotection from hydrogen exchange due to phosphorylation. Electrostatic attraction in the phosphorylated enzyme accounts for 1) domain 4 drawing closer to domains 1 and 3; 2) decreased accessibility; and 3) increased stability within these domains. The electrostriction due to phosphorylation may help capture substrate, whereas the opening of the cleft upon transient dephosphorylation allows rotation of the intermediate. The long-range effects of phosphorylation on hydrogen exchange parallel reports on protein kinases, suggesting a conceptual link among these multidomain, phosphoryl transfer enzymes.     

NMR resonance assignment of selectively labeled proteins by the use of paramagnetic ligands

Selective isotopic labeling of larger proteins greatly simplifies protein NMR spectra and reduces signal overlap, but selectively labeled proteins cannot be easily assigned since the sequential assignment method is not applicable. Here we describe a strategy for resonance assignment in selectively labeled proteins. Our approach involves a spin-labeled analog of a ligand of which the three-dimensional structure in complex with the target protein is known. Other methods for introduction of the spin label are possible. The paramagnetic center causes faster relaxation of all neighboring nuclei in a distance-dependent manner. Measurement of this effect allows to deduce distances between isotopically labeled residues and the paramagnetic center which can be used for resonance assignment. The method is demonstrated for the catalytic domain of Abl kinase in complex with the inhibitor, STI571.     

The first crystal structure of L-threonine dehydrogenase

L-threonine dehydrogenase (TDH) is an enzyme that catalyzes the oxidation of L-threonine to 2-amino-3-ketobutyrate. We solved the first crystal structure of a medium chain L-threonine dehydrogenase from a hyperthermophilic archaeon, Pyrococcus horikoshii (PhTDH), by the single wavelength anomalous diffraction method using a selenomethionine-substituted enzyme. This recombinant PhTDH is a homo-tetramer in solution. Three monomers of PhTDHs were located in the crystallographic asymmetric unit, however, the crystal structure exhibits a homo-tetramer structure with crystallographic and non-crystallographic 222 symmetry in the cell. Despite the low level of sequence identity to a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) and the different substrate specificity, the overall folds of the PhTDH monomer and tetramer are similar to those of the other ADH. Each subunit is composed of two domains: a nicotinamide cofactor (NAD(H))-binding domain and a catalytic domain. The NAD(H)-binding domain contains the alpha/beta Rossmann fold motif, characteristic of the NAD(H)-binding protein. One molecule of PhTDH contains one zinc ion playing a structural role. This metal ion exhibits coordination with four cysteine ligands and some of the ligands are conserved throughout the structural zinc-containing ADHs and TDHs. However, the catalytic zinc ion that is coordinated at the bottom of the cleft in the case of ADH was not observed in the crystal of PhTDH. There is a significant difference in the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft.     

Complete sequence-specific 1H NMR assignments for human insulin

Solvent conditions where human insulin could be studied by high-resolution NMR were determined. Both low pH and addition of acetonitrile were required to overcome the protein's self-association and to obtain useful spectra. Two hundred eighty-six 1H resonances were located and assigned to specific sites on the protein by using two-dimensional NMR methods. The presence and position of numerous dNN sequential NOE's indicate that the insulin conformation seen in crystallographic studies is largely retained under these solution conditions. Slowly exchanging protons were observed for seven backbone amide protons and were assigned to positions A15 and A16 and to positions B15-B19. These amides all occur within helical regions of the protein [Chawdhury, S.A., Dodson, E.J., Dodson, G.G., Reynolds, C.D., Tolley, S.P., Blundell, T.L., Cleasby, A., Pitts, J.E., Tickle, I.J., & Wood, S.P. (1983) Diabetologia 25, 460-464].     

Protein kinase A targeting and activation as seen by small-angle solution scattering

We have studied the solution structures of the multi-functional protein kinase A using small-angle X-ray and neutron scattering and have found a remarkable structural diversity in the different isoforms of this multi-subunit enzyme, in spite of its having high sequence homology and a common domain organization within its sequences. The available high-resolution crystal and NMR structural data for the protein kinase A components have aided in the interpretation of the solution scattering data and enabled us to develop models that bring insights into protein kinase A activation and targeting mechanisms, such as the opening and closing of the catalytic cleft to facilitate substrate binding or inhibition, respectively, and the role of sequence segments that join functional domains in the R subunit in providing a structurally flexible scaffold for interactions with the C subunit and A kinase-anchoring proteins (AKAPs).     

The solution structure of the N-terminal proteinase domain of the hepatitis C virus (HCV) NS3 protein provides new insights into its activation and catalytic mechanism.

The solution structure of the hepatitis C virus (BK strain) NS3 protein N-terminal domain (186 residues) has been solved by NMR spectroscopy. The protein is a serine protease with a chymotrypsin-type fold, and is involved in the maturation of the viral polyprotein. Despite the knowledge that its activity is enhanced by the action of a viral protein cofactor, NS4A, the mechanism of activation is not yet clear. The analysis of the folding in solution and the differences from the crystallographic structures allow the formulation of a model in which, in addition to the NS4A cofactor, the substrate plays an important role in the activation of the catalytic mechanism. A unique structural feature is the presence of a zinc-binding site exposed on the surface, subject to a slow conformational exchange process.     

The N-terminal domain of the Drosophila histone mRNA binding protein, SLBP, is intrinsically disordered with nascent helical structure

Stem-loop binding protein (SLBP) is a 31 kDa protein that is central to the regulation of histone mRNAs and is highly conserved in metazoans. In vertebrates, the N-terminal domain of SLBP has sequence determinants necessary for histone mRNA translation, SLBP degradation, cyclin binding, and histone mRNA import. We have used high-resolution NMR spectroscopy and circular dichroism to characterize the structural and dynamic features of this domain of SLBP from Drosophila (dSLBP). We report that the N-terminal domain of dSLBP is stably unfolded but has nascent helical structure at physiological pH and native-like solution conditions. The conformational and dynamic properties of the isolated domain are mimicked in a longer 175-residue region of the N-terminus, as well as in the full-length protein. Complete resonance assignments, secondary structure propensity, and motional properties of a 91-residue N-terminal domain (G17-K108) of dSLBP are reported here. The deviation of (1)H(alpha), (13)C(alpha), and (13)C(beta) chemical shifts from random coil reveals that there are four regions between residues I28-A45, S50-L57, S66-G75, and F91-N96 that have helical propensity. These regions also have small but positive heteronuclear NOEs, interresidue d(NN) NOEs, and small but significant protection from solvent exchange. However the lack of medium- and long-range NOEs in 3D (15)N- and (13)C-edited spectra, fast amide proton exchange rates (all greater than 1 s(-1)), and long (15)N relaxation (T(1), T(2)) times suggest that the domain from dSLBP does not adopt a well-defined tertiary fold. The backbone residual dipolar couplings (RDCs) for this domain are small and lie close to 0 Hz (+/-2 Hz) for most residues with no well-defined periodicity. The implications of this unfolded state for the function of dSLBP in regulating histone metabolism are discussed.     

Structural basis for matrix metalloproteinase-2 (MMP-2)-selective inhibitory action of β-amyloid precursor protein-derived inhibitor

Unlike other synthetic or physiological inhibitors for matrix metalloproteinases (MMPs), the β-amyloid precursor protein-derived inhibitory peptide (APP-IP) having an ISYGNDALMP sequence has a high selectivity toward MMP-2. Our previous study identified amino acid residues of MMP-2 essential for its selective inhibition by APP-IP and demonstrated that the N to C direction of the decapeptide inhibitor relative to the substrate-binding cleft of MMP-2 is opposite that of substrate. However, detailed interactions between the two molecules remained to be clarified. Here, we determined the crystal structure of the catalytic domain of MMP-2 in complex with APP-IP. We found that APP-IP in the complex is indeed embedded into the substrate-binding cleft of the catalytic domain in the N to C direction opposite that of substrate. With the crystal structure, it was first clarified that the aromatic side chain of Tyr(3) of the inhibitor is accommodated into the S1' pocket of the protease, and the carboxylate group of Asp(6) of APP-IP coordinates bidentately to the catalytic zinc of the enzyme. The Ala(7) to Pro(10) and Tyr(3) to Ile(1) strands of the inhibitor extend into the nonprime and the prime sides of the cleft, respectively. Therefore, the decapeptide inhibitor has long range contact with the substrate-binding cleft of the protease. This mode of interaction is probably essential for the high MMP-2 selectivity of the inhibitor because MMPs share a common architecture in the vicinity of the catalytic center, but whole structures of their substrate-binding clefts have sufficient variety for the inhibitor to distinguish MMP-2 from other MMPs.     

Crystal structure of the autocatalytic initiator of glycogen biosynthesis,glycogenin

The crystal structure of a medium-chain NAD(H)-dependent alcohol dehydrogenase (ADH) from an archaeon has been solved by multiwavelength anomalous diffraction, using a selenomethionine-substituted enzyme. The protein (SsADH), extracted from the hyperthermophilic organism Sulfolobus solfataricus, is a homo-tetramer with a crystallographic 222 symmetry. Despite the low level of sequence identity, the overall fold of the monomer is similar to that of the other homologous ADHs of known structure. However, a significant difference is the orientation of the catalytic domain relative to the coenzyme-binding domain that results in a larger interdomain cleft. At the bottom of this cleft, the catalytic zinc ion is coordinated tetrahedrally and lacks the zinc-bound water molecule that is usually found in ADH apoform structures. The fourth coordination position is indeed occupied by a Glu residue, as found in bacterial tetrameric ADHs. Other differences are found in the architecture of the substrate pocket whose entrance is more restricted than in other ADHs. SsADH is the first tetrameric ADH X-ray structure containing a second zinc ion playing a structural role. This latter metal ion shows a peculiar coordination, with a glutamic acid residue replacing one of the four cysteine ligands that are highly conserved throughout the structural zinc-containing dimeric ADHs.     

Determinants of function and substrate specificity in human UDP-galactose 4'-epimerase

UDP-galactose 4'-epimerase (GALE) interconverts UDP-galactose and UDP-glucose in the final step of the Leloir pathway. Unlike the Escherichia coli enzyme, human GALE (hGALE) also efficiently interconverts a larger pair of substrates: UDP-N-acetylgalactosamine and UDP-N-acetylglucosamine. The basis of this differential substrate specificity has remained obscure. Recently, however, x-ray crystallographic data have both predicted essential active site residues and suggested that differential active site cleft volume may be a key factor in determining GALE substrate selectivity. We report here a direct test of this hypothesis. In brief, we have created four substituted alleles: S132A, Y157F, S132A/Y157F, and C307Y-hGALE. While the first three substitutions were predicted to disrupt catalytic activity, the fourth was predicted to reduce active site cleft volume, thereby limiting entry or rotation of the larger but not the smaller substrate. All four alleles were expressed in a null-background strain of Saccharomyces cerevisiae and characterized in terms of activity with regard to both UDP-galactose and UDP-N-acetylgalactosamine. The S132A/Y157F and C307Y-hGALE proteins were also overexpressed in Pichia pastoris and purified for analysis. In all forms tested, the Y157F, S132A, and Y157F/S132A-hGALE proteins each demonstrated a complete loss of activity with respect to both substrates. In contrast, the C307Y-hGALE demonstrated normal activity with respect to UDP-galactose but complete loss of activity with respect to UDP-N-acetylgalactosamine. Together, these results serve to validate the wild-type hGALE crystal structure and fully support the hypothesis that residue 307 acts as a gatekeeper mediating substrate access to the hGALE active site.     

The protease associated (PA) domain in ScpA from Streptococcus pyogenes plays a role in substrate recruitment

Annually, over 18 million disease cases and half a million deaths worldwide are estimated to be caused by Group A Streptococcus. ScpA (or C5a peptidase) is a well characterised member of the cell enveleope protease family, which possess a S8 subtilisin-like catalytic domain and a shared multi-domain architecture. ScpA cleaves complement factors C5a and C3a, impairing the function of these critical anaphylatoxins and disrupts complement-mediated innate immunity. Although the high resolution structure of ScpA is known, the details of how it recognises its substrate are only just emerging. Previous studies have identified a distant exosite on the 2nd fibronectin domain that plays an important role in recruitment via an interaction with the substrate core. Here, using a combination of solution NMR spectroscopy, mutagenesis with functional assays and computational approaches we identify a second exosite within the protease-associated (PA) domain. We propose a model in which the PA domain assists optimal delivery of the substrate's C terminus to the active site for cleavage.     

Structural and dynamics characterization of norovirus protease

Norovirus protease is an essential enzyme for proteolytic maturation of norovirus nonstructural proteins and has been implicated as a potential target for antiviral drug development. Although X‐ray structural studies of the protease give us wealth of structural information including interactions of the protease with its substrate and dimeric overall structure, the role of protein dynamics in the substrate recognition and the biological relevance of the protease dimer remain unclear. Here we determined the solution NMR structure of the 3C‐like protease from Norwalk virus (NV 3CLpro), a prototype strain of norovirus, and analyzed its backbone dynamics and hydrodynamic behavior in solution. ¹⁵N spin relaxation and analytical ultracentrifugation analyses demonstrate that NV 3CLpro is predominantly a monomer in solution. Solution structure of NV 3CLpro shows significant structural variation in C‐terminal domain compared with crystal structures and among lower energy structure ensembles. Also, ¹⁵N spin relaxation and Carr–Purcell–Meiboom–Gill (CPMG)‐based relaxation dispersion analyses reveal the dynamic properties of residues in the C‐terminal domain over a wide range of timescales. In particular, the long loop spanning residues T123–G133 show fast motion (ps‐ns), and the residues in the bII–cII region forming the large hydrophobic pocket (S2 site) undergo conformational exchanges on slower timescales (μs–ms), suggesting their important role in substrate recognition.     

Structural characterization of the RNase E S1 domain and identification of its oligonucleotide-binding and dimerization interfaces

S1 domains occur in four of the major enzymes of mRNA decay in Escherichia coli: RNase E, PNPase, RNase II, and RNase G. Here, we report the structure of the S1 domain of RNase E, determined by both X-ray crystallography and NMR spectroscopy. The RNase E S1 domain adopts an OB-fold, very similar to that found with PNPase and the major cold shock proteins, in which flexible loops are appended to a well-ordered five-stranded beta-barrel core. Within the crystal lattice, the protein forms a dimer stabilized primarily by intermolecular hydrophobic packing. Consistent with this observation, light-scattering, chemical crosslinking, and NMR spectroscopic measurements confirm that the isolated RNase E S1 domain undergoes a specific monomer-dimer equilibrium in solution with a K(D) value in the millimolar range. The substitution of glycine 66 with serine dramatically destabilizes the folded structure of this domain, thereby providing an explanation for the temperature-sensitive phenotype associated with this mutation in full-length RNase E. Based on amide chemical shift perturbation mapping, the binding surface for a single-stranded DNA dodecamer (K(D)=160(+/-40)microM) was identified as a groove of positive electrostatic potential containing several exposed aromatic side-chains. This surface, which corresponds to the conserved ligand-binding cleft found in numerous OB-fold proteins, lies distal to the dimerization interface, such that two independent oligonucleotide-binding sites can exist in the dimeric form of the RNase E S1 domain. Based on these data, we propose that the S1 domain serves a dual role of dimerization to aid in the formation of the tetrameric quaternary structure of RNase E as described by Callaghan et al. in 2003 and of substrate binding to facilitate RNA hydrolysis by the adjacent catalytic domains within this multimeric enzyme.     

Molecular determinants of ligand specificity in family 11 carbohydrate binding modules: an NMR, X-ray crystallography and computational chemistry approach

The direct conversion of plant cell wall polysaccharides into soluble sugars is one of the most important reactions on earth, and is performed by certain microorganisms such as Clostridium thermocellum (Ct). These organisms produce extracellular multi-subunit complexes (i.e. cellulosomes) comprising a consortium of enzymes, which contain noncatalytic carbohydrate-binding modules (CBM) that increase the activity of the catalytic module. In the present study, we describe a combined approach by X-ray crystallography, NMR and computational chemistry that aimed to gain further insight into the binding mode of different carbohydrates (cellobiose, cellotetraose and cellohexaose) to the binding pocket of the family 11 CBM. The crystal structure of C. thermocellum CBM11 has been resolved to 1.98 A in the apo form. Since the structure with a bound substrate could not be obtained, computational studies with cellobiose, cellotetraose and cellohexaose were carried out to determine the molecular recognition of glucose polymers by CtCBM11. These studies revealed a specificity area at the CtCBM11 binding cleft, which is lined with several aspartate residues. In addition, a cluster of aromatic residues was found to be important for guiding and packing of the polysaccharide. The binding cleft of CtCBM11 interacts more strongly with the central glucose units of cellotetraose and cellohexaose, mainly through interactions with the sugar units at positions 2 and 6. This model of binding is supported by saturation transfer difference NMR experiments and linebroadening NMR studies.     

The three-dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76-Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1-hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three-dimensional structure of wild-type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R-factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five-stranded, mixed β-sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open-faced, anti-parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side-chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen-bonds and salt-bridges that are formed between its acetate and propionate side-groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side-chain of Asp84 hydrogen-bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring-coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three-dimensional structure and mechanism of activity. © 1996 Wiley-Liss, Inc.