Cysteine dioxygenase (CDO) catalyzes the oxidation of L-cysteine to cysteine sulfinic acid. Deficiencies in this enzyme have been linked to autoimmune diseases and neurological disorders. The x-ray crystal structure of CDO from Mus musculus was solved to a nominal resolution of 1.75 Å. The sequence is 91% identical to that of a human homolog. The structure reveals that CDO adopts the typical ␤-barrel fold of the cupin superfamily. The NE2 atoms of His-86, -88, and -140 provide the metal binding site. The structure further revealed a covalent linkage between the side chains of Cys-93 and Tyr-157, the cysteine of which is conserved only in eukaryotic proteins. Metal analysis showed that the recombinant enzyme contained a mixture of iron, nickel, and zinc, with increased iron content associated with increased catalytic activity. Details of the predicted active site are used to present and discuss a plausible mechanism of action for the enzyme.cupin ͉ cysteine metabolism ͉ O2-activation M ouse cysteine dioxygenase (CDO) catalyzes the initial step in the biochemical pathway used for oxidation of cysteine to sulfate (1), namely the oxidation of L-cysteine to cysteine sulfinic acid as shown in Fig. 1. The enzyme activity has important medical implications because elevated cysteine levels have been associated with Parkinson's and Alzheimer's diseases (2). High cysteine-to-sulfate ratios have been observed in patients suffering from systemic lupus erthematosus and rheumatoid arthritis (3, 4). Moreover, the Hallervorden-Spatz syndrome, a neurological disorder associated with iron accumulation, has been linked to a decline in CDO activity (5).CDO displays significant sequence identity with some members of the cupin superfamily (6), which have a conserved ␤-barrel fold and share two conserved sequence motifs: G(X) 5 HXH(X) 3,4 E(X) 6 G and G(X) 5 PXG(X) 2 H(X) 3 N (6-8).The two His and Glu residues from the first motif and the His from the second motif coordinate the metal ion in germin, the superfamily archetype (9). The Mus musculus CDO sequence contains the first motif with the exception of the glutamate, which is replaced by cysteine. This substitution is conserved in other eukaryotic CDOs. The second motif is less conserved, and only the His and Asn residues are present in the mouse CDO.CDO does not require an external reductant ( Fig. 1) and incorporates both oxygen atoms from O 2 (10), which justifies the dioxygenase classification, but relatively little else is known about the reaction mechanism. The recombinant enzyme from Rattus norvegicus has been purified and characterized by steadystate kinetics (11); the mouse enzyme investigated here has an identical sequence. Reconstitution of the rat apoenzyme with various transition metals confirmed that iron was required for activity, in accord with the earlier conclusions (1). Moreover, the recombinant rat enzyme was active without a second interacting factor, despite previous reports suggesting that additional components were required (12, 13).Here, we describe the x-ra...

initiation ͉ transcription ͉ wrapping ͉ kinetics S pecific transcription initiation by Escherichia coli RNA polymerase (RNAP: core subunit composition ␣ 2 ␤␤Ј ϩ 70 ϭ holoenzyme) at promoter sequences is determined by recognition of DNA (Ϫ10 and Ϫ35 hexamers) upstream of the start site (ϩ1) by the specificity subunit 70 . Subsequent to binding, a series of large-scale conformational changes in both RNAP and promoter DNA create the initiation-competent open complex (RP o ) (1). During these steps, the multisubunit bacterial RNAP acts as an intricate molecular machine and opens Ϸ14 bp of the DNA double helix. Defining the cascade of conformational changes that occur during initiation is essential to understand sequence-and factordependent regulation of the rate of transcription initiation and has important applications in chemical biology and in antibiotic design. However, the intermediates on this pathway are relatively unstable and short-lived and hence are difficult to trap unambiguously. To date, all structural information about complexes known to be on-pathway intermediates in RP o formation has come from chemical and enzymatic DNA footprinting methods.Quantitative kinetic-mechanistic studies find that at least two kinetically significant intermediates, generically designated I 1 and I 2 , precede formation of RP o by E. coli RNAP:where the relatively slow interconversions between I 1 and I 2 are rate-limiting in both the forward and back directions (2, 3). In the mechanism shown in Eq. 1, I 2 and RP o are characterized by their resistance to a short challenge with a polyanionic competitor such as heparin, which acts to sequester any free RNAP present during the challenge. (In contrast, after a 10 to 20 sec challenge with heparin, I 1 complexes, which are in rapid equilibrium with free RNAP and promoter sequences, are eliminated from the population.) Given the high degree of conservation of bacterial RNAP and promoter DNA sequences, this mechanism is likely to describe the key steps in initiation in most prokaryotes. Moreover, conservation of many elements of sequence, structure, and/or function between bacterial and eukaryotic polymerase (pol II) subunits and transcription factors supports the inference that the bacterial intermediates may be homologs of initiation intermediates formed by pol II (4, 5).Recently we (6) and Ross and Gourse (7) found that the presence of DNA upstream of the Ϫ35 promoter recognition hexamer greatly accelerates (up to Ϸ60-fold) the rate-determining isomerization step (conversion of I 1 to I 2 ). Strikingly, DNase I footprinting of I 1 at the strong bacteriophage promoter P R reveals that when nonspecific DNA upstream of base pair Ϫ47 is present, downstream DNA is protected to around ϩ20, and thus bound in the active-site channel of RNAP. However, when DNA upstream of Ϫ47 is deleted,

The ribonuclease inhibitor protein (RI) binds to members of the bovine pancreatic ribonuclease (RNase A) superfamily with an affinity in the femtomolar range. Here, we report on structural and energetic aspects of the interaction between human RI (hRI) and human pancreatic ribonuclease (RNase 1). The structure of the crystalline hRI·RNase 1 complex was determined at a resolution of 1.95 Å, revealing the formation of 19 intermolecular hydrogen bonds involving 13 residues of RNase 1. In contrast, only 9 such hydrogen bonds are apparent in the structure of the complex between porcine RI and RNase A. hRI, which is anionic, also appears to use its horseshoe-shaped structure to engender long-range Coulombic interactions with RNase 1, which is cationic. In accordance with the structural data, the hRI·RNase 1 complex was found to be extremely stable (t ½ = 81 days; K d = 2.9 × 10 -16 M). Site-directed mutagenesis experiments enabled the identification of two cationic residues in RNase 1-Arg39 and Arg91-that are especially important for both the formation and stability of the complex, and are thus termed "electrostatic targeting residues". Disturbing the electrostatic attraction between hRI and RNase 1 yielded a variant of RNase 1 that maintained ribonucleolytic activity and conformational stability but had a 2.8 × 10 3 -fold lower association rate for complex formation and 5.9 × 10 9 -fold lower affinity for hRI. This variant of RNase 1, which exhibits the largest decrease in RI affinity of any engineered ribonuclease, is also toxic to human erythroleukemia cells. Together, these results provide new insight into an unusual and important protein-protein interaction, and could expedite the development of human ribonucleases as chemotherapeutic agents.

SUMMARY The ancient UbiB protein kinase-like family is involved in isoprenoid lipid biosynthesis and is implicated in human diseases, but demonstration of UbiB kinase activity has remained elusive for unknown reasons. Here, we quantitatively define UbiB-specific sequence motifs and reveal their positions within the crystal structure of a UbiB protein, ADCK3. We find that multiple UbiB-specific features are poised to inhibit protein kinase activity, including an N-terminal domain that occupies the typical substrate binding pocket and a unique A-rich loop that limits ATP binding by establishing an unusual selectivity for ADP. A single alanine-to-glycine mutation of this loop flips this coenzyme selectivity and enables autophosphorylation, but inhibits coenzyme Q biosynthesis in vivo, demonstrating functional relevance for this unique feature. Our work provides mechanistic insight into UbiB enzyme activity and establishes a molecular foundation for further investigation of how UbiB family proteins affect diseases and diverse biological pathways.

SUMMARY The UbiB protein kinase-like (PKL) family is widespread—comprising one-quarter of microbial PKLs and five human homologs—yet its biochemical activities remain obscure. COQ8A (ADCK3) is a mammalian UbiB protein associated with ubiquinone (CoQ) biosynthesis and an ataxia (ARCA2) through unclear means. We show that mice lacking COQ8A develop a slowly progressive cerebellar ataxia linked to Purkinje cell dysfunction and mild exercise intolerance, recapitulating ARCA2. Interspecies biochemical analyses show that COQ8A and yeast Coq8p specifically stabilize a CoQ biosynthesis complex through unorthodox PKL functions. While COQ8 was predicted to be a protein kinase, we demonstrate that it lacks canonical protein kinase activity in trans. Instead, COQ8 has ATPase activity and interacts with lipid CoQ intermediates—functions that are likely conserved across all domains of life. Collectively, our results lend insight into the molecular activities of the ancient UbiB family and elucidate the biochemical underpinnings of a human disease.