Determination of the structure of seleno-methionine-labelled hydroxymethylbilane synthase in its active form by multi-wavelength anomalous dispersion.

The enzyme hydroxymethylbilane synthase (HMBS, E.C. 4.3.1.8) catalyzes the conversion of porphobilinogen into hydroxymethylbilane, a key intermediate for the biosynthesis of heme, chlorophylls, vitamin B12 and related macrocycles. The enzyme is found in all organisms, except viruses. The crystal structure of the selenomethionine-labelled enzyme ([SeMet]HMBS) from Escherichia coli has been solved by the multi-wavelength anomalous dispersion (MAD) experimental method using the Daresbury SRS station 9.5. In addition, [SeMet]HMBS has been studied by MAD at the Grenoble ESRF MAD beamline BM14 (BL19) and this work is described especially with respect to the use of the ESRF CCD detector. The structure at ambient temperature has been refined, the R factor being 16.8% at 2. 4 A resolution. The dipyrromethane cofactor of the enzyme is preserved in its reduced form in the crystal and its geometrical shape is in full agreement with the crystal structures of authentic dipyrromethanes. Proximal to the reactive C atom of the reduced cofactor, spherical density is seen consistent with there being a water molecule ideally placed to take part in the final step of the enzyme reaction cycle. Intriguingly, the loop with residues 47-58 is not ordered in the structure of this form of the enzyme, which carries no substrate. Direct experimental study of the active enzyme is now feasible using time-resolved Laue diffraction and freeze-trapping, building on the structural work described here as the foundation.

Biosynthesis of vitamin B12: the multi-enzyme synthesis of precorrin-4 and factor IV.

BACKGROUND: In order to study the biosynthesis of vitamin B12, it is necessary to produce various intermediates along the biosynthetic pathway by enzymic methods. Recently, information on the organisation of the biosynthetic pathway has permitted the selection of the set of enzymes needed to biosynthesise any specific identified intermediate. The aim of the present work was to use recombinant enzymes in reconstituted multi-enzyme systems to biosynthesise particular intermediates. RESULTS: The products of the cobG and cobJ genes from Pseudomonas denitrificans were expressed heterologously in Escherichia coli to afford good levels of activity of the corresponding enzymes, CobG and CobJ. Aerobic incubation of precorrin-3A with the CobG enzyme alone yielded precorrin-3B. When CobJ and S-adenosyl-L-methionine were included in the incubation, the product was precorrin-4. Both precorrin 3B and precorrin-4 are known precursors of vitamin B12 and their availability has allowed new mechanistic studies of enzymic transformations. CONCLUSIONS: Our results show that the expression of the CobG and CobJ enzymes has been successful, thus facilitating the biosynthesis of two precursors of vitamin B12. This lays the foundation for the structure determination of CobG and CobJ as well as future enzymic experiments focusing on later steps of vitamin B12 biosynthesis.

Biosynthesis of vitamin B12: the preparative multi-enzyme synthesis of precorrin-3A and 20-methylsirohydrochlorin (a 2,7,20-trimethylisobacteriochlorin).

The Bacillus subtilis genes hemB, hemC and hemD, encoding respectively the enzymes porphobilinogen synthase, hydroxymethylbilane synthase and uroporphyrinogen III synthase, have been expressed in Escherichia coli using a single plasmid construct. An enzyme preparation from this source converts 5-aminolaevulinic acid (ALA) preparatively and in high yield into uroporphyrinogen III. The Pseudomonas denitrificans genes cobA and cobI, encoding respectively the enzymes S-adenosyl-L-methionine:uroporphyrinogen III methyltransferase (SUMT) and S-adenosyl-L-methionine:precorrin-2 methyltransferase (SP2MT), were also expressed in E. coli. When SUMT was combined with the coupled-enzyme system that produces uroporphyrinogen III, precorrin-2 was synthesized from ALA, and when SP2MT was also added the product from the coupling of five enzymes was precorrin-3A. Both of these products are precursors of vitamin B12, and they can be used directly for biosynthetic experiments or isolated as their didehydro octamethyl esters in > 40% overall yield. The enzyme system which produces precorrin-3A is sufficiently stable to allow long incubations on a large scale, affording substantial quantities (15-20 mg) of product.

Biosynthesis of porphyrins and related macrocycles, Part 43. Isolation and characterization of intermediates of coenzyme B12 biosynthesis, a cobyrinic acid triamide, the a,c-diamide and their Co-(5'-deoxy-5'-adenosyl) derivatives, from Propionibacterium shermanii.

BACKGROUND: Vitamin B12 is synthesized by many different organisms, for example Pseudomonas denitrificans (aerobic) and Propionibacterium shermanii ('microaerophilic', or essentially anaerobic). The biosynthetic pathways in these two organisms show strong similarities but also some differences. There have been conflicting reports on where differences between these two organisms lie in the stages beyond the formation of the corrin macrocycle. Characterization of intermediates in the pathway will help resolve these conflicts. RESULTS: A single cobyrinic acid diamide and a single triamide have been isolated from Pr. shermanii. The diamide was shown to be the a,c-isomer. The triamide is not the a,c,g-isomer but it is indistinguishable from the single triamide isolated by other workers from Ps. denitrificans. The Co-(5'-deoxy-5'-adenosyl) derivative of the a,c-diamide was also isolated and fully characterized and the deoxyadenosyl derivative of the foregoing triamide has been shown to be present in the cells. CONCLUSIONS: Our results support a unique pathway in Pr. shermanii proceeding from cobyrinic acid towards coenzyme B12, at least as far as the adenosylated triamide intermediate. No evidence was found for multiple alternative pathways. The order of amidations of the carboxyl side-chains of cobyrinic acid up to the triamide stage is the same in Pr. shermanii and Ps. denitrificans.

Expression, purification and characterisation of the product from the Bacillus subtilis hemD gene, uroporphyrinogen III synthase.

Uroporphyrinogen III synthase, the product of the hemD gene, is the enzyme responsible for the cyclisation of the linear tetrapyrrole, hydroxymethylbilane. The hemD gene isolated from Bacillus subtilis was manipulated by PCR to enable direct cloning behind a synthetic ribosome-binding site downstream of tandem bacteriophage lambda PR and PL promoters in a pCE30-derived vector. Following thermal induction of transcription, the resulting plasmid (pPS21) directed the synthesis of uroporphyrinogen III synthase. The protein produced was soluble and was readily purified. Pure uroporphyrinogen III synthase is monomeric with an isoelectric point of 4.1 and an optimum pH for activity of 8.3. Its specific activity by assay using synthetic hydroxymethylbilane as substrate is 565 units mg-1 and the Km for this substrate is 330 +/- 30 nM. The N-terminal sequence of the enzyme is Met-Glu-Asn-Asp-Phe-Pro-Leu, in agreement with the gene-derived sequence. Studies based on amino acid modifications suggest that arginine, lysine and probably histidine residues are essential for the activity of uroporphyrinogen III synthase. Significantly, this synthase from B. subtilis is substantially more thermostable than the enzymes from previously studied sources.

How nature builds the pigments of life: the conquest of vitamin B12.

In part because humans cannot synthesize vitamin B12 and must obtain it from organisms that produce it and because B12 deficiency leads to pernicious anemia, it has been important to understand how microorganisms build this quite complex substance. As shown here, an interdisciplinary attack was needed, which combined the strengths of genetics, molecular biology, enzymology, chemistry, and spectroscopy. This allowed the step-by-step synthetic pathway of B12 to be elucidated, and this approach has acted as a model for future research on the synthesis of substances in living organisms. One practical outcome of such an approach has been the improved availability of B12 for animal feedstuffs and human health.

New intermediates in the B12 pathway.

Vitamin B12 has a complex structure which represents one of the most challenging biosynthetic problems in Nature. Exciting progress has been made by combining the techniques, approaches and strengths of chemistry, spectroscopy and biology. Most of the advances until recently came from experiments based either on labelling simpler precursors with radioactive isotopes followed by controlled degradation of the labelled products, or on the use of stable isotopes, 13C in particular, because it can be detected and its environment can be studied by NMR spectroscopy. These experiments imposed heavy demands on synthesis which provided the specifically labelled starting materials. More recently, the powerful methods of genetics and molecular biology have been added to the armoury, leading to another massive surge forward by allowing the preparation, through gene overexpression, of large quantities of the enzymes of the biosynthetic pathway. Equally important has been the generation of mutant forms of B12-producing organisms in which the biosynthetic pathway is blocked at specific points. Here I focus on the latest advances. The structures of the newly discovered intermediates are described and some of the chemistry involved is explored. In conclusion, the presently known pathway to vitamin B12 is reviewed.

Purification, characterization, crystallisation and X-ray analysis of selenomethionine-labelled hydroxymethylbilane synthase from Escherichia coli.

Hydroxymethylbilane synthase (HMBS) catalyses the conversion of porphobilinogen into hydroxymethylbilane, a linear tetrapyrrolic intermediate in the biosynthesis of haems, chlorophylls, vitamin B12 and related macrocycles. In the course of an investigation of the crystal structure of this enzyme, we intended to follow a new strategy to obtain the X-ray phase information, i.e. the collection of multiwavelength anomalous diffraction data from a crystal of a seleno-L-methionine (SeMet)-labelled variant of the protein. We have expressed and purified HMBS from Escherichia coli (34268 Da) in which all (six) methionine (Met) residues are replaced by SeMet. Complete replacement, as shown by amino acid composition analysis and by electrospray mass spectrometry, was achieved by growing the Met-requiring mutant E. coli PO1562 carrying the plasmid pPA410 in a medium containing 50 mg/l SeMet as the sole source of Met. [SeMet]HMBS exhibits full enzyme activity, as reflected by unchanged steady-state kinetic parameters relative to native enzyme. Rhombohedral crystals of [SeMet]HMBS could be grown at the pH optimum (7.4) of the enzyme (solutions containing 30 mg/ml protein, 0.4 mM EDTA, 20 mM dithiothreitol, 3 M NaCl and 15 mM Bristris-propane buffer were equilibrated by vapour diffusion at 20 degrees C against reservoirs of saturated NaCl). However, being very thin plates, these crystals were not suitable for X-ray analysis. Alternatively, rectangular crystals were obtained at pH 5.3 using conditions based on those reported for wild-type HMBS [sitting drops of 50 microliters containing 6-7 mg/ml protein, 0.3 mM EDTA, 15 mM dithiothreitol, 10% (mass/vol.) poly(ethylene glycol) 6000 and 0.01% NaN3 in 0.1 M sodium acetate were equilibrated by vapour diffusion at 20 degrees C against a reservoir of 10-20 mg solid dithiothreitol]. X-ray diffraction data of the crystals were complete to 93.8% at 0.21 nm resolution and showed that [SeMet]HMBS and native HMBS crystallise isomorphously. A difference Fourier map using FSeMet-Fnative and phases derived from the native structure, which has recently been determined independently by multiple isomorphous replacement, showed positive difference peaks centered at or close to where the sulphur atoms of the Met side chains appear in the native structure. In addition, paired positive/negative peaks in the difference map near the cofactor of HMBS indicate conformational differences in the active site, probably due to differences in the state of oxidation of the cofactor in the two crystalline samples.

Studies on the mechanism of hydroxymethylbilane synthase concerning the role of arginine residues in substrate binding.

The role of conserved arginine residues in hydroxymethylbilane synthase was investigated by replacing these residues in the enzyme from Escherichia coli with leucine residues by using site-directed mutagenesis. The kinetic parameters for these mutant enzymes and studies on the formation of intermediate enzyme-substrate complexes indicate that several of these arginine residues are involved in binding the carboxylate side chains of the pyrromethane cofactor and the growing oligopyrrole chain.

Biosynthesis of vitamin B12: structure of precorrin-6x octamethyl ester.

13C-labeled precorrin-6x is biosynthesized by cell-free protein preparations from Pseudomonas denitrificans in separate experiments using delta-amino[5-13C]levulinic acid and the corresponding delta-amino[4-13C]- and delta-amino[3-13C]levulinic acid-labeled forms in conjunction with S-[methyl-13C]adenosylmethionine for the latter two experiments. These labeled precorrin-6x samples, as their octamethyl esters, are studied by a range of NMR techniques. In addition, nuclear Overhauser effect difference measurements are made on unlabeled precorrin-6x ester to determine connectivities. The structure 6a so established for precorrin-6x ester (i) confirms the results reported in the preceding paper that precorrin-6x has a ring-contracted macrocycle, still carries the C-12 acetate residue, and stands at the oxidation level of a dehydrocorrin; (ii) reveals the unexpected methylation at C-11 not C-12, leading to a structure with separated chromophores; and (iii) implies that methyl migration from C-11 to C-12 occurs when precorrin-6x is converted into hydrogenobyrinic acid. Proposals for the biosynthesis of the corrin macrocycle of hydrogenobyrinic acid and vitamin B12 are made.

Investigation of putative active-site lysine residues in hydroxymethylbilane synthase. Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine.

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold over-expression of hydroxymethylbilane synthase (HMBS). This construct was used to generate HMBS in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 were replaced by glutamine (K55Q, K59Q and K55Q-K59Q respectively). All three modified enzymes are chromatographically separable from wild-type enzyme. Kinetic studies showed that the substitution K55Q has little effect whereas K59Q causes a 25-fold decrease in Kapp. cat./Kapp. m. Treatment of K55Q, K59Q and K55Q-K59Q separately with pyridoxal 5'-phosphate and NaBH4 resulted in incomplete and non-specific reaction with the remaining lysine residues. Pyridoxal modification of Lys-59 in the K55Q mutant caused greater enzymic inactivation than similar modification of Lys-55 in K59Q. The results in sum show that, though Lys-55 and Lys-59 may be at or near the active site, neither is indispensable for the catalytic activity of HMBS.

Evidence that pyridoxal phosphate modification of lysine residues (Lys-55 and Lys-59) causes inactivation of hydroxymethylbilane synthase (porphobilinogen deaminase).

A recombinant strain of Escherichia coli has been constructed that produces approx. 200 times the amount of hydroxymethylbilane synthase found in wild-type E. coli [Hart, Abell & Battersby (1986) Biochem. J. 240, 273-276]. Enzyme purified from this strain is shown to be permanently inactivated by pyridoxal 5'-phosphate/NaB1H3(3)H1. The inactivation is not complete despite the fact that approx. 1 mol of lysine residues is modified per mol of enzyme. Evidence is gained showing that (a) modification of one of two conserved lysine residues (Lys-55 or Lys-59) results in inactivation of hydroxymethylbilane synthase and (b) these lysine residues are present in or close to the active site.

Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound to the protein through the sulphur atom of cysteine-242.

The pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli is bound to the protein through the sulphur atom of a cysteine residue [Hart, Miller & Battersby (1988) Biochem. J. 252, 909-912; Beifuss, Hart, Miller & Battersby (1988) Tetrahedron Lett. 29, 2591-2594]. We show that the pyrromethane-binding residue is cysteine-242.

Biosynthesis of the pigments of life.

Chlorophyll can be chosen as the fundamental pigment of life. Related to it is protoheme, the red pigment of blood. These substances are found to be biosynthesized from the same parent substance called uroporphyrinogen-III. This paper covers research over a period of 19 years, which has gradually revealed the details of Nature's pathway to uroporphyrinogen-III.

Synthetic and biosynthetic studies on vitamin B12.

Vitamin B12 is the anti-pernicious anemia vitamin. It functions as the main part of a coenzyme for a range of remarkable rearrangement reactions. The macrocycle for vitamin B12 is smaller by one carbon than that for chlorophyll and protoheme. Research is described involving numerous skills at the microscale level. In addition, synthesis of new pigments that have been encountered has been undertaken. A large part of the biosynthetic pathway to vitamin B12 has been revealed.

Evidence that the pyrromethane cofactor of hydroxymethylbilane synthase (porphobilinogen deaminase) is bound through the sulphur atom of a cysteine residue.

Hydroxymethylbilane synthase (porphobilinogen deaminase) from Escherichia coli uses a novel pyrromethane cofactor to bind the growing pyrrolic chain for hydroxymethylbilane biosynthesis [Hart, Miller, Leeper & Battersby (1987) J. Chem. Soc. Chem. Commun. 1762-1765]. We show that this cofactor is bound to the protein through the sulphur atom of a cysteine residue.