Biochemical analysis of a dimerization domain mutation in RetGC-1 associated with dominant cone-rod dystrophy.

Mutations in the photoreceptor membrane guanylyl cyclase RetGC-1 have been linked to autosomal dominant cone-rod dystrophy. Three mutations were identified that alter strictly conserved residues within the RetGC-1 dimerization domain, a region predicted to form an amphipathic alpha-helical coil. Here we report on a biochemical characterization of one of the mutations, a substitution of cysteine for arginine at residue 838. We generated this mutation in vitro and measured its catalytic activity and sensitivity to guanylyl cyclase activating protein 1 (GCAP-1) and GCAP-2. The R838C substitution has several effects. It reduces the overall catalytic ability of RetGC-1 and dramatically reduces stimulation by GCAP-2, although GCAP-2 still appears to interact with the protein. The R838C substitution also increases the apparent affinity of RetGC-1 for GCAP-1 and alters the Ca(2+) sensitivity of the GCAP-1 response, allowing the mutant to be stimulated by GCAP-1 at higher Ca(2+) concentrations than wild type. The diminished response to GCAP-2, which we propose is not likely the cause of cone-rod degeneration in these patients, is interesting mechanistically because it separates the ability to bind a specific GCAP from the ability to be stimulated by it, and it also discriminates between the mechanisms of activation of GCAP-1 vs. GCAP-2. We suggest that the gain-of-function effects of R838C on RetGC-1 stimulated by GCAP-1, which are dominant in vitro and may cause an abnormal increase in cGMP synthesis in dark-adapted photoreceptors, may be the cause of the cone-rod degeneration.

Cobalamin (vitamin B12) biosynthesis--cloning, expression and crystallisation of the Bacillus megaterium S-adenosyl-L-methionine-dependent cobalt-precorrin-4 transmethylase CbiF.

The Bacillus megaterium cbiF, encoding the cobalt-precorrin-4 S-adenosyl-L-methionine-dependent transmethylase of the anaerobic cobalamin biosynthetic pathway, has been cloned and overexpressed as a His-tagged recombinant protein in Escherichia coli. The protein was purified to homogeneity by a combination of metal chelate chromatography and high-resolution anion-exchange chromatography. The protein migrated with a subunit mass of 31 kDa by SDS/PAGE and with a molecular mass of 62 kDa by analytical gel filtration, suggesting that the native recombinant protein is a homodimer. The His-tagged protein was physiologically active as it was able to complement a Salmonella typhimurium cbiF mutant. However, the protein did not bind S-adenosyl-L-methionine with the same avidity as observed with other corrin biosynthetic transmethylases. A crystallisation screen of the purified protein led to the identification of two discrete crystal forms. One of these forms has been characterised and a full data set collected.

The X-ray structure of a cobalamin biosynthetic enzyme, cobalt-precorrin-4 methyltransferase.

Biosynthesis of the corrin ring of vitamin B12 requires the action of six S-adenosyl-L-methionine (AdoMet) dependent transmethylases, closely related in sequence. The first X-ray structure of one of these, cobalt-precorrin-4 transmethylase, CbiF, from Bacillus megaterium has been determined to a resolution of 2.4 A. CbiF contains two alphabeta domains forming a trough in which S-adenosyl-L-homocysteine (AdoHcy) binds. The location of AdoHcy and a number of conserved residues, helps define the precorrin binding site. A second crystal form determined at 3.1 A resolution highlights the flexibility of two loops around this site. CbiF employs a unique mode of AdoHcy binding and represents a new class of transmethylase.

Effect of mutations in the transmethylase and dehydrogenase/chelatase domains of sirohaem synthase (CysG) on sirohaem and cobalamin biosynthesis.

The Escherichia coli CysG protein (sirohaem synthase) catalyses four separate reactions that are required for the transformation of uroporphyrinogen III into sirohaem, initially two S-adenosyl-l-methionine-dependent transmethylations at positions 2 and 7, mediated through the C-terminal, or CysGA, catalytic domain of the protein, and subsequently a ferrochelation and dehydrogenation, mediated through the N-terminal, or CysGB, catalytic domain of the enzyme. This report describes how the deletion of the NAD+-binding site of CysG, located within the first 35 residues of the N-terminus, is detrimental to the activity of CysGB but does not affect the catalytic activity of CysGA, whereas the mutation of a number of phylogenetically conserved residues within CysGA is detrimental to the transmethylation reaction but does not affect the activity of CysGB. Further studies have shown that CysGB is not essential for cobalamin biosynthesis because the presence of the Salmonella typhimurium CobI operon with either cysGA or the Pseudomonas denitrificans cobA are sufficient for the synthesis of cobyric acid in an E. coli cysG deletion strain. Evidence is also presented to suggest that a gene within the S. typhimurium CobI operon might act as a chelatase that, at low levels of cobalt, is able to aid in the synthesis of sirohaem.

Siroheme biosynthesis in higher plants. Analysis of an S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferase from Arabidopsis thaliana.

Siroheme, the prosthetic group for both nitrite and sulfite reductases, is a methylated, iron-containing modified tetrapyrrole. Here we report the first molecular characterization of the branch point enzyme in higher plants, which directs intermediates toward siroheme synthesis. A cDNA was cloned from Arabidopsis thaliana (UPM1) that functionally complements an Escherichia coli cysG mutant, a strain that is unable to catalyze the conversion of uroporphyrinogen III (Uro'gen-III) to siroheme. UPM1 is 1484 base pairs and encodes a 369-amino acid, 39.9-kDa protein. The UPM1 product contains two regions that are identical to consensus sequences found in bacterial Uro'gen-III and precorrin methyltransferases. Recombinant UPM1 protein was found to catalyze S-adenosyl-L-methionine-dependent transmethylation by UPM1 in a multistep process involving the formation of a covalently linked complex with S-adenosyl-L-methionine. The UPM1 product has a sequence at the amino terminus that resembles a transit peptide for localization to mitochondria or plastids. The protein produced by in vitro expression is able to enter isolated intact chloroplasts but not mitochondria. Genomic blot analysis showed that UPM1 is encoded in the A. thaliana genome. The genomic DNA corresponding to UPM1 was cloned and sequenced and found to contain at least five introns.

Evidence for a covalent intermediate in the S-adenosyl-L-methionine-dependent transmethylation reaction catalysed by sirohaem synthase.

CysG, also known as uroporphyrinogen III methylase and sirohaem synthase (CysG; EC 2.1.1.107), is a multifunctional enzyme that is able to transform uroporphyrinogen III into sirohaem via two S-adenosyl-L-methionine (AdoMet)-dependent transmethylations, an NAD(+)-dependent dehydrogenation and a ferrochelation. The apparent tight binding of AdoMet to this multifunctional enzyme is investigated. The use of a rapid AdoMet binding assay demonstrates that CysG becomes labelled with both [methyl-3H]AdoMet and [carboxyl-14C]AdoMet. Further experiments show that the CysG-AdoMet complex is subsequently able to methylate uroporphyrinogen III. CysG remains associated with the labelled constituents of the AdoMet even after denaturation with urea and SDS/PAGE, suggesting that the AdoMet has become covalently linked to the protein. A rapid examination of some of the other transmethylases involved in corrin biosynthesis reveals that they bind the AdoMet in a similar fashion. A multistep transmethylation mechanism is proposed to explain the observed results.

Gene dissection demonstrates that the Escherichia coli cysG gene encodes a multifunctional protein.

The C-terminus of the Escherichia coli CysG protein, consisting of amino acids 202-457, was expressed as a recombinant protein using gene dissection methodology. Analysis of the activity of this truncated protein, termed CysGA, revealed that it was able to methylate uroporphyrinogen III in the same S-adenosyl-L-methionine (SAM)-dependent manner as the complete CysG protein. However, this truncated protein was not able to complement E. coli cysG cells, thereby suggesting that the first 201 amino acids of the CysG protein had an enzymic activity associated with the conversion of dihydrosirohydrochlorin into sirohaem. Analysis of the N-terminus of the CysG protein revealed the presence of a putative pyridine dinucleotide binding site. When the purified CysG protein was incubated with NADP+, uroporphyrinogen III and SAM the enzyme was found to catalyse a coenzyme-mediated dehydrogenation to form sirohydrochlorin. The CysGA protein on the other hand showed no such coenzyme-dependent activity. Analysis of the porphyrinoid material isolated from strains harbouring plasmids containing the complete and truncated cysG genes suggested that the CysG protein was also involved in ferrochelation. The evidence presented in this paper suggests that the CysG protein is a multifunctional protein involved in SAM-dependent methylation, pyridine dinucleotide dependent dehydrogenation and ferrochelation.

Evidence for participation of aspartate-84 as a catalytic group at the active site of porphobilinogen deaminase obtained by site-directed mutagenesis of the hemC gene from Escherichia coli.

The role of aspartate-84, an invariant residue in the active site cleft of Escherichia coli porphobilinogen deaminase, has been investigated by site-directed mutagenesis. Substitution of aspartate-84 by glutamate results in an enzyme that retains less than 1% of its activity and which can form highly stable enzyme-intermediate complexes. Substitution of aspartate-84 by either alanine or asparagine, however, results in proteins unable to catalyze the formation of preuroporphyrinogen but which, nevertheless, appear able to assemble the dipyrromethane cofactor. The mechanisms of the tetramerization reaction and cofactor assembly are discussed.

5-Aminolaevulinic acid synthase and uroporphyrinogen methylase: two key control enzymes of tetrapyrrole biosynthesis and modification.

Two enzymes which play an important role in regulation and flux control through the tetapyrrole biosynthetic pathway are considered. The Rhodobacter sphaeroides 5-aminolaevulinic acid synthase isoenzymes are discussed and the progress being made on their recombinant expression and isolation is reported. The Escherichia coli uroporphyrinogen methylase, which is encoded by the cysG gene, is also examined. In this case evidence is provided which demonstrates that the gene product is responsible for the complete synthesis of sirohaem from uroporphyrinogen III. The enzyme is thus capable of performing two S-adenosylmethionine-dependent methylation reactions, an NADP(+)-dependent dehydrogenation and iron chelation. The uroporphyrinogen methylase is thus a small multifunctional enzyme.

Structural studies on porphobilinogen deaminase.

The X-ray crystallographic analysis of porphobilinogen deaminase (hydroxymethylbilane synthase, EC 4.3.1.8) shows the polypeptide chain folded into three domains, (1) N-terminal, (2) central and (3) C-terminal, of approximately equal size. Domains 1 and 2 have a similar overall topology, a modified doubly wound parallel beta-sheet. Domain 3 is an open-faced three-stranded antiparallel beta-sheet, with one face covered by three alpha-helices. The active site is located between domains 1 and 2. The dipyrromethane cofactor linked to cysteine 242 protrudes from domain 3 into the mouth of the cleft. Flexible segments between domains 1 and 2 are thought to have a role in a hinge mechanism, facilitating conformational changes. The cleft is lined with positively charged, highly conserved, arginine residues which form ion pairs with the acidic side chains of the cofactor. Aspartic acid 84 has been identified as a critical catalytic residue both by its proximity to the cofactor pyrrole ring nitrogen and by structural and kinetic studies of the Asp-84-->Glu mutant protein. The active site arginine residues have been altered by site-directed mutagenesis to histidine residues. The mutant proteins have been studied crystallographically in order to reconcile the functional changes in the polymerization reaction with structural changes in the enzyme.

Structure of porphobilinogen deaminase reveals a flexible multidomain polymerase with a single catalytic site.

The three-domain structure of porphobilinogen deaminase, a key enzyme in the biosynthetic pathway of tetrapyrroles, has been defined by X-ray analysis at 1.9 A resolution. Two of the domains structurally resemble the transferrins and periplasmic binding proteins. The dipyrromethane cofactor is covalently linked to domain 3 but is bound by extensive salt-bridges and hydrogen-bonds within the cleft between domains 1 and 2, at a position corresponding to the binding sites for small-molecule ligands in the analogous proteins. The X-ray structure and results from site-directed mutagenesis provide evidence for a single catalytic site. Interdomain flexibility may aid elongation of the polypyrrole product in the active-site cleft of the enzyme.

Mutagenesis of arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase that affects dipyrromethane cofactor assembly and tetrapyrrole chain initiation and elongation.

Substitutions of conserved arginine residues in the catalytic cleft of Escherichia coli porphobilinogen deaminase were constructed by site-specific mutagenesis of the hemC gene. Mutant proteins exhibited a range of defects including the failure to assemble the dipyrromethane cofactor and the inability to initiate and propagate the tetrapolymerization reaction. Mutations of arginine residues at positions 11, 131, 132 and 155, all of which interact with the carboxylic acid side chains of the dipyrromethane cofactor, were the most disruptive.