Substrate Shuttling between Active Sites of Uroporphyrinogen Decarboxylase Is Not Required to Generate Coproporphyrinogen

Uroporphyrinogen decarboxylase (URO-D; EC 4.1.1.37), the fifth enzyme of the heme biosynthetic pathway, is required for the production of heme, vitamin B12, siroheme, and chlorophyll precursors. URO-D catalyzes the sequential decarboxylation of four acetate side chains in the pyrrole groups of uroporphyrinogen to produce coproporphyrinogen. URO-D is a stable homodimer, with the active-site clefts of the two subunits adjacent to each other. It has been hypothesized that the two catalytic centers interact functionally, perhaps by shuttling of reaction intermediates between subunits. We tested this hypothesis by construction of a single-chain protein (single-chain URO-D) in which the two subunits were connected by a flexible linker. The crystal structure of this protein was shown to be superimposable with wild-type activity and to have comparable catalytic activity. Mutations that impaired one or the other of the two active sites of single-chain URO-D resulted in approximately half of wild-type activity. The distributions of reaction intermediates were the same for mutant and wild-type sequences and were unaltered in a competition experiment using I and III isomer substrates. These observations indicate that communication between active sites is not required for enzyme function and suggest that the dimeric structure of URO-D is required to achieve conformational stability and to create a large active-site cleft.     

Structural insights into the processivity of endopolygalacturonase I from Aspergillus niger

Endopolygalacturonase I is a processive enzyme, while the 60% sequence identical endopolygalacturonase II is not. The 1.70 A resolution crystal structure of endopolygalacturonase I reveals a narrowed substrate binding cleft. In addition, Arg96, a residue in this cleft previously shown to be critical for processivity, interacts with the substrate mimics glycerol and sulfate in several well-defined conformations in the six molecules in the asymmetric unit. From this we conclude that both Arg96 and the narrowed substrate binding cleft contribute to retaining the substrate while it moves through the active site after a cleavage event has occurred.     

Three-dimensional structure of 2-amino-3-ketobutyrate CoA ligase from Escherichia coli complexed with a PLP-substrate intermediate: inferred reaction mechanism

2-Amino-3-ketobutyrate CoA ligase (KBL, EC 2.3.1.29) is a pyridoxal phosphate (PLP) dependent enzyme, which catalyzes the second reaction step on the main metabolic degradation pathway for threonine. It acts in concert with threonine dehydrogenase and converts 2-amino-3-ketobutyrate, the product of threonine dehydrogenation by the latter enzyme, with the participation of cofactor CoA, to glycine and acetyl-CoA. The enzyme has been well conserved during evolution, with 54% amino acid sequence identity between the Escherichia coli and human enzymes. We present the three-dimensional structure of E. coli KBL determined at 2.0 A resolution. KBL belongs to the alpha family of PLP-dependent enzymes, for which the prototypic member is aspartate aminotransferase. Its closest structural homologue is E. coli 8-amino-7-oxononanoate synthase. Like many other members of the alpha family, the functional form of KBL is a dimer, and one such dimer is found in the asymmetric unit in the crystal. There are two active sites per dimer, located at the dimer interface. Both monomers contribute side chains to each active/substrate binding site. Electron density maps indicated the presence in the crystal of the Schiff base intermediate of 2-amino-3-ketobutyrate and PLP, an external aldimine, which remained bound to KBL throughout the protein purification procedure. The observed interactions between the aldimine and the side chains in the substrate binding site explain the specificity for the substrate and provide the basis for a detailed proposal of the reaction mechanism of KBL. A putative binding site of the CoA cofactor was assigned, and implications for the cooperation with threonine dehydrogenase were considered.     

Modeling and alanine scanning mutagenesis studies of recombinant pokeweed antiviral protein

The Phytolacca americana-derived naturally occurring ribosome inhibitory protein pokeweed antiviral protein (PAP) is an N-glycosidase that catalytically removes a specific adenine residue from the stem loop of ribosomal RNA. We have employed molecular modeling studies using a novel model of PAP-RNA complexes and site-directed mutagenesis combined with bioassays to evaluate the importance of the residues at the catalytic site and a putative RNA binding active center cleft between the catalytic site and C-terminal domain for the enzymatic deadenylation of ribosomal RNA by PAP. As anticipated, alanine substitutions by site-directed mutagenesis of the PAP active site residues Tyr(72), Tyr(123), Glu(176), and Arg(179) that directly participate in the catalytic deadenylation of RNA resulted in greater than 3 logs of loss in depurinating and ribosome inhibitory activity. Similarly, alanine substitution of the conserved active site residue Trp(208), which results in the loss of stabilizing hydrophobic interactions with the ribose as well as a hydrogen bond to the phosphate backbone of the RNA substrate, caused greater than 3 logs of loss in enzymatic activity. By comparison, alanine substitutions of residues (28)KD(29), (80)FE(81), (111)SR(112), (166)FL(167) that are distant from the active site did not significantly reduce the enzymatic activity of PAP. Our modeling studies predicted that the residues of the active center cleft could via electrostatic interactions contribute to both the correct orientation and stable binding of the substrate RNA molecule in the active site pocket. Notably, alanine substitutions of the highly conserved, charged, and polar residues of the active site cleft including (48)KY(49), (67)RR(68), (69)NN(70), and (90)FND(92) substantially reduced the depurinating and ribosome inhibitory activity of PAP. These results provide unprecedented evidence that besides the active site residues of PAP, the conserved, charged, and polar side chains located at its active center cleft also play a critical role in the PAP-mediated depurination of ribosomal RNA.     

Crystal structures of Escherichia coli uridine phosphorylase in two native and three complexed forms reveal basis of substrate specificity, induced conformational changes and influence of potassium

Uridine phosphorylase (UP) is a key enzyme in the pyrimidine salvage pathway that catalyses the reversible phosphorolysis of uridine to uracil and ribose 1-phosphate. Inhibiting liver UP in humans raises blood uridine levels and produces a protective effect ("uridine rescue") against the toxicity of the chemotherapeutic agent 5-fluorouracil without reducing its antitumour activity. We have investigated UP-substrate interactions by determining the crystal structures of native Escherichia coli UP (two forms), and complexes with 5-fluorouracil/ribose 1-phosphate, 2-deoxyuridine/phosphate and thymidine/phosphate. These hexameric structures confirm the overall structural similarity of UP to E.coli purine nucleoside phosphorylase (PNP) whereby, in the presence of substrate, each displays a closed conformation resulting from a concerted movement that closes the active site cleft. However, in contrast to PNP where helix segmentation is the major conformational change between the open and closed forms, in UP more extensive changes are observed. In particular a swinging movement of a flap region consisting of residues 224-234 seals the active site. This overall change in conformation results in compression of the active site cleft. Gln166 and Arg168, part of an inserted segment not seen in PNP, are key residues in the uracil binding pocket and together with a tightly bound water molecule are seen to be involved in the substrate specificity of UP. Enzyme activity shows a twofold dependence on potassium ion concentration. The presence of a potassium ion at the monomer/monomer interface induces some local rearrangement, which results in dimer stabilisation. The conservation of key residues and interactions with substrate in the phosphate and ribose binding pockets suggest that ribooxocarbenium ion formation during catalysis of UP may be similar to that proposed for E.coli PNP.     

An allosteric circuit in caspase-1

Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k_cat/K_m). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k_cat/K_m. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling.     

The structure of aconitase

The crystal structure of the 80,000 Da Fe-S enzyme aconitase has been solved and refined at 2.1 A resolution. The protein contains four domains; the first three from the N-terminus are closely associated around the [3Fe-4S] cluster with all three cysteine ligands to the cluster being provided by the third domain. Association of the larger C-terminal domain with the first three domains creates an extensive cleft leading to the Fe-S cluster. Residues from all four domains contribute to the active site region, which is defined by the Fe-S cluster and a bound SO4(2-) ion. This region of the structure contains 4 Arg, 3 His, 3 Ser, 2 Asp, 1 Glu, 3 Asn, and 1 Gln residues, as well as several bound water molecules. Three of these side chains reside on a three-turn 3(10) helix in the first domain. The SO4(2-) ion is bound 9.3 A from the center of the [3Fe-4S] cluster by the side chains of 2 Arg and 1 Gln residues. Each of 3 His side chains in the putative active site is paired with Asp or Glu side chains.     

Oligosaccharide binding to barley alpha-amylase 1

Enzymatic subsite mapping earlier predicted 10 binding subsites in the active site substrate binding cleft of barley alpha-amylase isozymes. The three-dimensional structures of the oligosaccharide complexes with barley alpha-amylase isozyme 1 (AMY1) described here give for the first time a thorough insight into the substrate binding by describing residues defining 9 subsites, namely -7 through +2. These structures support that the pseudotetrasaccharide inhibitor acarbose is hydrolyzed by the active enzymes. Moreover, sugar binding was observed to the starch granule-binding site previously determined in barley alpha-amylase isozyme 2 (AMY2), and the sugar binding modes are compared between the two isozymes. The "sugar tongs" surface binding site discovered in the AMY1-thio-DP4 complex is confirmed in the present work. A site that putatively serves as an entrance for the substrate to the active site was proposed at the glycone part of the binding cleft, and the crystal structures of the catalytic nucleophile mutant (AMY1D180A) complexed with acarbose and maltoheptaose, respectively, suggest an additional role for the nucleophile in the stabilization of the Michaelis complex. Furthermore, probable roles are outlined for the surface binding sites. Our data support a model in which the two surface sites in AMY1 can interact with amylose chains in their naturally folded form. Because of the specificities of these two sites, they may locate/orient the enzyme in order to facilitate access to the active site for polysaccharide chains. Moreover, the sugar tongs surface site could also perform the unraveling of amylose chains, with the aid of Tyr-380 acting as "molecular tweezers."     

Crystal structure of Methanobacterium thermoautotrophicum phosphoribosyl-AMP cyclohydrolase HisI

The metabolic pathway for histidine biosynthesis is interesting from an evolutionary perspective because of the diversity of gene organizations and protein structures involved. Hydrolysis of phosphoribosyl-AMP, the third step in the histidine biosynthetic pathway, is carried out by PR-AMP cyclohydrolase, the product of the hisI gene. The three-dimensional structure of PR-AMP cyclohydrolase from Methanobacterium thermoautotrophicum was solved and refined to 1.7 A resolution. The enzyme is a homodimer. The position of the Zn(2+)-binding site that is essential for catalysis was inferred from the positions of bound Cd(2+) ions, which were part of the crystallization medium. These metal binding sites include three cysteine ligands, two from one monomer and the third from the second monomer. The enzyme remains active when Cd(2+) is substituted for Zn(2+). The likely binding site for Mg(2+), also necessary for activity in a homologous cyclohydrolase, was also inferred from Cd(2+) positions and is comprised of aspartic acid side chains. The putative substrate-binding cleft is formed at the interface between the two monomers of the dimer. This fact, combined with the localization of the Zn(2+)-binding site, indicates that the enzyme is an obligate dimer.     

26 kDa endochitinase from barley seeds: an interaction of the ionizable side chains essential for catalysis

To explore the structure essential for the catalysis in 26 kDa endochitinase from barley seeds, we calculated theoretical pKa values of the ionizable groups based on the crystal structure, and then the roles of ionizable side chains located near the catalytic residue were examined by site-directed mutagenesis. The pKa value calculated for Arg215, which is located at the bottom of the catalytic cleft, is abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. No enzymatic activity was found in the Arg215-mutated chitinase (R215A) produced by the Escherichia coli expression system. The transition temperature of thermal unfolding (T(m)) of R215A was lower than that of the wild type protein by about 6.2 degrees C. In the crystal structure, the Arg215 side chain is in close proximity to the Glu203 side chain, whose theoretical pKa value was found to be abnormally low (-2.4), suggesting that these side chains may interact with each other. Mutation of Glu203 to alanine (E203A) completely eliminated the enzymatic activity and impaired the thermal stability (deltaT(m) = 6.4 degrees C) of the enzyme. Substrate binding ability was also affected by the Glu203 mutation. These data clearly demonstrate that the Arg215 side chain interacts with the Glu203 side chain to stabilize the conformation of the catalytic cleft. A similar interaction network was previously found in chitosanase from Streptomyces sp. N174 [Fukamizo et al. (2000) J. Biol. Chem. 275, 25633-25640]; hence, this type of interaction seems to be at least partly conserved in the catalytic cleft of other glycosyl hydrolases.     

Binding interactions between the active center cleft of recombinant pokeweed antiviral protein and the alpha-sarcin/ricin stem loop of ribosomal RNA

Pokeweed antiviral protein (PAP) is a ribosome-inactivating protein that catalytically cleaves a specific adenine base from the highly conserved alpha-sarcin/ricin loop of the large ribosomal RNA, thereby inhibiting protein synthesis at the elongation step. Recently, we discovered that alanine substitutions of the active center cleft residues significantly impair the depurinating and ribosome inhibitory activity of PAP. Here we employed site-directed mutagenesis combined with standard filter binding assays, equilibrium binding assays with Scatchard analyses, and surface plasmon resonance technology to elucidate the putative role of the PAP active center cleft in the binding of PAP to the alpha-sarcin/ricin stem loop of rRNA. Our findings presented herein provide experimental evidence that besides the catalytic site, the active center cleft also participates in the binding of PAP to the target tetraloop structure of rRNA. These results extend our recent modeling studies, which predicted that the residues of the active center cleft could, via electrostatic interactions, contribute to both the correct orientation and stable binding of the substrate RNA molecules in PAP active site pocket. The insights gained from this study also explain why and how the conserved charged and polar side chains located at the active center cleft of PAP and certain catalytic site residues, that do not directly participate in the catalytic deadenylation of ribosomal RNA, play a critical role in the catalytic removal of the adenine base from target rRNA substrates by affecting the binding interactions between PAP and rRNA.     

Characterization and crystallization of human uroporphyrinogen decarboxylase.

The cytosolic enzyme uroporphyrinogen decarboxylase (URO-D) catalyzes the fifth step in the heme biosynthetic pathway, converting uroporphyrinogen to coproporphyrinogen by decarboxylating the four acetate side chains of the substrate. Recombinant human URO-D has been expressed in Escherichia coli with a histidine tag and has been purified to homogeneity. Purified protein was determined to be a monodisperse dimer by dynamic light scattering. Equilibrium sedimentation analysis confirmed that the protein is dimeric, with a dissociation constant of 0.1 microM. URO-D containing an amino-terminal histidine tag was crystallized in space group P3(1)21 or its enantiomer P3(2)21 with unit cell dimensions a = b = 103.6 A, c = 75.2 A. There is one molecule in the asymmetric unit, consistent with generation of the dimer by the twofold axis of this crystallographic operator. Native data have been collected to 3.0 a resolution.     

Structural origins of amino acid selection without editing by cysteinyl-tRNA synthetase

Cysteinyl-tRNA synthetase (CysRS) is highly specific for synthesis of cysteinyl adenylate, yet does not possess the amino acid editing activity characteristic of many other tRNA synthetases. To elucidate how CysRS is able to distinguish cysteine from non-cognate amino acids, crystal structures of the Escherichia coli enzyme were determined in apo and cysteine-bound states. The structures reveal that the substrate cysteine thiolate forms a single direct interaction with a zinc ion bound at the base of the active site cleft, in a trigonal bipyramidal geometry together with four highly conserved protein side chains. Cysteine binding induces movement of the zinc ion towards substrate, as well as flipping of the conserved Trp205 indole ring to pack on the thiol side chain. The imidazole groups of five conserved histidines lie adjacent to the zinc ion, forming a unique arrangement suggestive of functional significance. Thus, amino acid discrimination without editing arises most directly from the favorable zinc-thiolate interaction, which is not possible for non-cognate substrates. Additional selectivity may be generated during the induced-fit conformational changes that help assemble the active site.     

The crystal structure of yeast thiamin pyrophosphokinase.

BACKGROUND: Thiamin pyrophosphokinase (TPK) catalyzes the transfer of a pyrophosphate group from ATP to vitamin B1 (thiamin) to form the coenzyme thiamin pyrophosphate (TPP). Thus, TPK is important for the formation of a coenzyme required for central metabolic functions. TPK has no sequence homologs in the PDB and functions by an unknown mechanism. The TPK structure has been determined as a significant step toward elucidating its catalytic action. RESULTS: The crystal structure of Saccharomyces cerevisiae TPK complexed with thiamin has been determined at 1.8 A resolution. TPK is a homodimer, and each subunit consists of two domains. One domain resembles a Rossman fold with four alpha helices on each side of a 6 strand parallel beta sheet. The other domain has one 4 strand and one 6 strand antiparallel beta sheet, which form a flattened sandwich structure containing a jelly-roll topology. The active site is located in a cleft at the dimer interface and is formed from residues from domains of both subunits. The TPK dimer contains two compound active sites at the subunit interface. CONCLUSIONS: The structure of TPK with one substrate bound identifies the location of the thiamin binding site and probable catalytic residues. The structure also suggests a likely binding site for ATP. These findings are further supported by TPK sequence homologies. Although possessing no significant sequence homology with other pyrophospokinases, thiamin pyrophosphokinase may operate by a mechanism of pyrophosphoryl transfer similar to those described for pyrophosphokinases functioning in nucleotide biosynthesis.     

A molecular dynamics investigation of mono and dimeric states of the outer membrane enzyme OMPLA

OMPLA is a phospholipase found in the outer membranes of many Gram-negative bacteria. Enzyme activation requires calcium-induced dimerisation plus bilayer perturbation. As the conformation of OMPLA in the different crystal forms (monomer versus dimer; with/without bound Ca(2+)) is remarkably similar we have used multi-nanosecond molecular dynamics (MD) simulations to probe possible differences in conformational dynamics that may be related to enzyme activation. Simulations of calcium-free monomeric OMPLA, of the Ca(2+)-bound dimer, and of the Ca(2+)-bound dimer with a substrate analogue covalently linked to the active site serine have been performed, all with the protein embedded in a phospholipid (POPC) bilayer. All simulations were stable, but differences in the dynamic behaviour of the protein between the various states were observed. In particular, the stability of the active site and the hydrophobic substrate-binding cleft varied. Dimeric OMPLA is less flexible than monomeric OMPLA, especially around the active site. In the absence of bound substrate analogue, the hydrophobic substrate-binding cleft of dimeric OMPLA collapses. A model is proposed whereby the increased stability of the active site in dimeric OMPLA is a consequence of the local ordering of water around the nearby calcium ion. The observed collapse of the substrate-binding cleft may explain the experimentally observed occurrence of multiple dimer conformations of OMPLA, one of which is fully active while the other shows significantly reduced activity.     

Probing the roles of active site residues in the 3'-5' exonuclease of the Werner syndrome protein

Werner syndrome is a premature aging disease caused by mutations in the WS gene and a deficiency in the function of Werner protein (WRN). The lack of WRN results in a cellular phenotype of genomic instability. WRN belongs to the RecQ DNA helicase family, but unlike other RecQ family members it possesses a functional exonuclease domain. We determined the crystal structure of mWRNexo (residues 31-238) bound to Zn(2+) and the sulfate ion. Compared with the structure of human WRNexo (hWRNexo), notable conformational changes were observed in several active site residues in an H5-H6 loop and in helices H6 and H7 of mWRNexo, presumably because of the presence of sulfate, which mimics the phosphate of substrate DNA. In particular, the side chains of Lys(185) and Tyr(206) were reoriented toward the Zn(2+) ion, whereas the side chain of Arg(190) pointed away from the active site center. Mutational analysis of these conserved residues abolished WRN exonuclease activity, suggesting that these residues play a critical role in the WRNexo activity. Based on substrate modeling and mutational analyses, we propose a mechanism by which WRNexo becomes activated upon substrate DNA binding. We also describe the low resolution trimeric structure of mouse WRNexoL (mWRNexoL, residues 31-330), as elucidated by small angle x-ray scattering (SAXS) analyses.     

The effect of mutations surrounding and within the active site on the catalytic activity of ricin A chain.

Models for the binding of the sarcin-ricin loop (SRL) of 28S ribosomal RNA to ricin A chain (RTA) suggest that several surface exposed arginine residues surrounding the active site cleft make important interactions with the RNA substrate. The data presented in this study suggest differing roles for these arginyl residues. Substitution of Arg48 or Arg213 with Ala lowered the activity of RTA 10-fold. Furthermore, substitution of Arg213 with Asp lowered the activity of RTA 100-fold. The crystal structure of this RTA variant showed it to have an unaltered tertiary structure, suggesting that the positively charged state of Arg213 is crucial for activity. Substitution of Arg258 with Ala had no effect on activity, although substitution with Asp lowered activity 10-fold. Substitution of Arg134 prevented expression of folded protein, suggesting a structural role for this residue. Several models have been proposed for the binding of the SRL to the active site of RTA in which the principal difference lies in the conformation of the second 'G' in the target GAGA motif in the 28S rRNA substrate. In one model, the sidechain of Asn122 is proposed to make interactions with this G, whereas another model proposes interactions with Asp75 and Asn78. Site-directed mutagenesis of these residues of RTA favours the first of these models, as substitution of Asn78 with Ser yielded an RTA variant whose activity was essentially wild-type, whereas substitution of Asn122 reduced activity 37.5-fold. Substitution of Asp75 failed to yield significant folded protein, suggesting a structural role for this residue.     

Regulated Cleavage of Prothrombin by Prothrombinase: REPOSITIONING A CLEAVAGE SITE REVEALS THE UNIQUE KINETIC BEHAVIOR OF THE ACTION OF PROTHROMBINASE ON ITS COMPOUND SUBSTRATE*?

Prothrombinase converts prothrombin to thrombin via cleavage at Arg³²⁰ followed by cleavage at Arg²⁷¹. Exosite-dependent binding of prothrombin to prothrombinase facilitates active site docking by Arg³²⁰ and initial cleavage at this site. Precise positioning of the Arg³²⁰ site for cleavage is implied by essentially normal cleavage at Arg³²⁰ in recombinant prothrombin variants bearing additional Arg side chains either one or two residues away. However, mutation of Arg³²⁰ to Gln reveals that prothrombinase can cleave prothrombin following Arg side chains shifted by as many as two residues N-terminal to the 320 position at near normal rates. Further repositioning leads to a loss in cleavage at this region with an abrupt shift toward slow cleavage at Arg²⁷¹. In contrast, the binding constant for the active site docking step is strongly dependent on the sequence preceding the scissile bond as well as position. Large effects on binding only yield minor changes in rate until the binding constant passes a threshold value. This behavior is expected for a substrate that can engage the enzyme through mutually exclusive active site docking reactions followed by cleavage to yield different products. Cleavage site specificity as well as the ordered action of prothrombinase on its compound substrate is regulated by the thermodynamics of active site engagement of the individual sites as well as competition between alternate cleavage sites for active site docking.     

Crystal structure of the priming beta-ketosynthase from the R1128 polyketide biosynthetic pathway

ZhuH is a priming ketosynthase that initiates the elongation of the polyketide chain in the biosynthetic pathway of a type II polyketide, R1128. The crystal structure of ZhuH in complex with the priming substrate acetyl-CoA reveals an extensive loop region at the dimer interface that appears to affect the selectivity for the primer unit. Acetyl-CoA is bound in a 20 A-long channel, which placed the acetyl group against the catalytic triad. Analysis of the primer unit binding site in ZhuH suggests that it can accommodate acyl chains that are two to four carbons long. Selectivity and primer unit size appear to involve the side chains of three residues on the loops close to the dimer interface that constitute the bottom of the substrate binding pocket.     

The crystal structures of chloramphenicol phosphotransferase reveal a novel inactivation mechanism

Chloramphenicol (Cm), produced by the soil bacterium Streptomyces venezuelae, is an inhibitor of bacterial ribosomal peptidyltransferase activity. The Cm-producing streptomycete modifies the primary (C-3) hydroxyl of the antibiotic by a novel Cm-inactivating enzyme, chloramphenicol 3-O-phosphotransferase (CPT). Here we describe the crystal structures of CPT in the absence and presence of bound substrates. The enzyme is dimeric in a sulfate-free solution and tetramerization is induced by ammonium sulfate, the crystallization precipitant. The tetrameric quaternary structure exhibits crystallographic 222 symmetry and has ATP binding pockets located at a crystallographic 2-fold axis. Steric hindrance allows only one ATP to bind per dimer within the tetramer. In addition to active site binding by Cm, an electron-dense feature resembling the enzyme's product is found at the other subunit interface. The structures of CPT suggest that an aspartate acts as a general base to accept a proton from the 3-hydroxyl of Cm, concurrent with nucleophilic attack of the resulting oxyanion on the gamma-phosphate of ATP. Comparison between liganded and substrate-free CPT structures highlights side chain movements of the active site's Arg136 guanidinium group of >9 A upon substrate binding.