Biochemical evidence for the tyrosine involvement in cationic intermediate stabilization in mouse β-carotene 15, 15'-monooxygenase

<p>Abstract</p> <p>Background</p> <p>β-carotene 15,15'-monooxygenase (BCMO1) catalyzes the crucial first step in vitamin A biosynthesis in animals. We wished to explore the possibility that a carbocation intermediate is formed during the cleavage reaction of BCMO1, as is seen for many isoprenoid biosynthesis enzymes, and to determine which residues in the substrate binding cleft are necessary for catalytic and substrate binding activity. To test this hypothesis, we replaced substrate cleft aromatic and acidic residues by site-directed mutagenesis. Enzymatic activity was measured <it>in vitro </it>using His-tag purified proteins and <it>in vivo </it>in a β-carotene-accumulating <it>E. coli </it>system.</p> <p>Results</p> <p>Our assays show that mutation of either Y235 or Y326 to leucine (no cation-π stabilization) significantly impairs the catalytic activity of the enzyme. Moreover, mutation of Y326 to glutamine (predicted to destabilize a putative carbocation) almost eliminates activity (9.3% of wt activity). However, replacement of these same tyrosines with phenylalanine or tryptophan does not significantly impair activity, indicating that aromaticity at these residues is crucial. Mutations of two other aromatic residues in the binding cleft of BCMO1, F51 and W454, to either another aromatic residue or to leucine do not influence the catalytic activity of the enzyme. Our <it>ab initio </it>model of BCMO1 with β-carotene mounted supports a mechanism involving cation-π stabilization by Y235 and Y326.</p> <p>Conclusions</p> <p>Our data are consistent with the formation of a substrate carbocation intermediate and cation-π stabilization of this intermediate by two aromatic residues in the substrate-binding cleft of BCMO1.</p

Structural analysis of the regulatory protein of pyrimidine biosynthesis, PyrR

Bacteria have evolved diverse mechanisms to control gene expression at the level of transcription and translation. Transcriptional attenuation of the genes of bacterial operons allows control of the extent of transcriptional readthrough in response to environmental signals. In Bacillus species, the expression of genes of the pyr operon, responsible for pyrimidine biosynthesis, is controlled by the product of the first gene of the operon, PyrR. In response to the availability of the uridylate end-product of the pathway, PyrR binds to the elongating mRNA transcript of the operon, controlling the formation of alternate RNA stem-loop structures that terminate further transcription. By virtue of its sequence and structural fold, PyrR belongs to the phosphoribosyltransferase (PRT) class of proteins. Although the protein displays some PRTase activity at elevated pH, under physiological conditions the regulatory, RNA-binding function of PyrR appears to be its primary role. Understanding the molecular basis for the evolution of the regulatory role of PyrR has been the goal of this project. Crystal structures of Bacillus caldolyticus PyrR provide insights into how this regulatory role might be achieved. For the first time, complexes of PyrR with the nucleotides, uridine monophosphate (UMP) and guanosine monophosphate (GMP), and divalent magnesium were obtained. Although the binding of UMP was expected, the association of GMP was a novel finding. The PyrR-nucleotide complexes support a model for dual regulation of PyrR by pyrimidines and purines. Sedimentation velocity experiments suggest that the stoichiometry of PyrR-RNA complex formation is one RNA molecule to one PyrR dimer. Electrophoretic mobility shift assays demonstrate that UMP and GMP have opposite effects on RNA binding to the PyrR dimer. Initial crystals of the PyrR-RNA complex, grown in presence of magnesium and UMP, have been obtained. Based on the structural and biochemical data reported here, as well as precedents from other PRT and transcriptional antitermination proteins, a structural model for the PyrR-RNA complex, as well as the role of regulatory nucleotides, is proposed. Overall, the results with the B. caldolyticus PyrR have increased our understanding of the unique adaptation of a PRTase enzyme for a novel RNA-binding role

Stabilizing interactions in the dimer interface of α-subunit in Escherichia coli RNA polymerase: A graph spectral and point mutation study

The formation of α2 dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the α-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the α-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the α-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli α-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the α-dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants α-F35A and α-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the α-NTD

Stabilizing interactions in the dimer interface of \alpha-subunit in Escherichia coli RNA polymerase: A graph spectral and point mutation study

The formation of $\alpha_2$ dimer in Escherichia coli core RNA polymerase (RNAP) is thought to be the first step toward the assembly of the functional enzyme. A large number of evidences indicate that the \alpha-subunit dimerizes through its N-terminal domain (NTD). The crystal structures of the \alpha-subunit NTD and that of a homologous Thermus aquaticus core RNAP are known. To identify the stabilizing interactions in the dimer interface of the \alpha-NTD of E. coli RNAP, we identified side-chain clusters by using the crystal structure coordinates of E. coli \alpha-NTD. A graph spectral algorithm was used to identify side-chain clusters. This algorithm considers the global nonbonded side-chain interactions of the residues for the clustering procedure and is unique in identifying residues that make the largest number of interactions among the residues that form clusters in a very quantitative way. By using this algorithm, a nine-residue cluster consisting of polar and hydrophobic residues was identified in the subunit interface adjacent to the hydrophobic core. The residues forming the cluster are relatively rigid regions of the interface, as measured by the thermal factors of the residues. Most of the cluster residues in the E. coli enzyme were topologically and sequentially conserved in the T. aquaticus RNAP crystal structure. Residues 35F and 46I were predicted to be important in the stability of the -\alpha dimer interface, with 35F forming the center of the cluster. The predictions were tested by isolating single-point mutants \alpha-F35A and \alpha-I46S on the dimer interface, which were found to disrupt dimerization. Thus, the identified cluster at the edge of the dimer interface seems to be a vital component in stabilizing the \alpha-NTD

Structure of the Nucleotide Complex of PyrR, the pyr Attenuation Protein from Bacillus caldolyticus, Suggests Dual Regulation by Pyrimidine and Purine Nucleotides

PyrR is a protein that regulates the expression of genes and operons of pyrimidine nucleotide biosynthesis (pyr genes) in many bacteria. PyrR acts by binding to specific sequences on pyr mRNA and causing transcriptional attenuation when intracellular levels of uridine nucleotides are elevated. PyrR from Bacillus subtilis has been purified and extensively studied. In this work, we describe the purification to homogeneity and characterization of recombinant PyrR from the thermophile Bacillus caldolyticus and the crystal structures of unliganded PyrR and a PyrR-nucleotide complex. The B. caldolyticus pyrR gene was previously shown to restore normal regulation of the B. subtilis pyr operon in a pyrR deletion mutant. Like B. subtilis PyrR, B. caldolyticus PyrR catalyzes the uracil phosphoribosyltransferase reaction but with maximal activity at 60°C. Crystal structures of B. caldolyticus PyrR reveal a dimer similar to the B. subtilis PyrR dimer and, for the first time, binding sites for nucleotides. UMP and GMP, accompanied by Mg(2+), bind specifically to PyrR active sites. Nucleotide binding to PyrR is similar to other phosphoribosyltransferases, but Mg(2+) binding differs. GMP binding was unexpected. The protein bound specific sequences of pyr RNA 100 to 1,000 times more tightly than B. subtilis PyrR, depending on the RNA tested and the assay method; uridine nucleotides enhanced RNA binding, but guanosine nucleotides antagonized it. The new findings of specific GMP binding and its antagonism of RNA binding suggest cross-regulation of the pyr operon by purines

RPE65, Visual Cycle Retinol Isomerase, Is Not Inherently 11-cis-specific: SUPPORT FOR A CARBOCATION MECHANISM OF RETINOL ISOMERIZATION*

The mechanism of retinol isomerization in the vertebrate retina visual cycle remains controversial. Does the isomerase enzyme RPE65 operate via nucleophilic addition at C11 of the all-trans substrate, or via a carbocation mechanism? To determine this, we modeled the RPE65 substrate cleft to identify residues interacting with substrate and/or intermediate. We find that wild-type RPE65 in vitro produces 13-cis and 11-cis isomers equally robustly. All Tyr-239 mutations abolish activity. Trp-331 mutations reduce activity (W331Y to ∼75% of wild type, W331F to ∼50%, and W331L and W331Q to 0%) establishing a requirement for aromaticity, consistent with cation-π carbocation stabilization. Two cleft residues modulate isomerization specificity: Thr-147 is important, because replacement by Ser increases 11-cis relative to 13-cis by 40% compared with wild type. Phe-103 mutations are opposite in action: F103L and F103I dramatically reduce 11-cis synthesis relative to 13-cis synthesis compared with wild type. Thr-147 and Phe-103 thus may be pivotal in controlling RPE65 specificity. Also, mutations affecting RPE65 activity coordinately depress 11-cis and 13-cis isomer production but diverge as 11-cis decreases to zero, whereas 13-cis reaches a plateau consistent with thermal isomerization. Lastly, experiments using labeled retinol showed exchange at 13-cis-retinol C15 oxygen, thus confirming enzymatic isomerization for both isomers. Thus, RPE65 is not inherently 11-cis-specific and can produce both 11- and 13-cis isomers, supporting a carbocation (or radical cation) mechanism for isomerization. Specific visual cycle selectivity for 11-cis isomers instead resides downstream, attributable to mass action by CRALBP, retinol dehydrogenase 5, and high affinity of opsin apoproteins for 11-cis-retinal