Peter M. Shoolingin-Jordan

Acute intermittent porphyria in Sweden. Molecular, functional and clinical consequences of some new mutations found in the porphobilinogen deaminase gene

Acute intermittent porphyria (AIP) is an autosomal dominant disorder caused by a partial deficit of porphobilinogen deaminase (PBGD), the third of eight enzymes in the haem biosynthetic pathway. The overt disease is characterized by neuropsychiatric symptoms that are often triggered by exogenous factors such as certain drugs, stress, and alcohol. The aim of this work has been to identify the underlying genetic defect in each AIP‐affected family in order to provide early counselling to assist in the avoidance of precipitating factors. The prevalence of AIP in Sweden is in the order of 1:10 000. The major mutation in Sweden, W198X, is due to a founder effect in the northern part of the country. This mutation, together with a further 11 mutations, have been reported previously. The present communication encompasses the great majority of AIP kindreds in Sweden and includes a further 27 mutations within the PBGD gene. This includes 14 completely new mutations, as well as 11 known mutations detected for the first time in Sweden. The majority of the mutations are located in exons 10 and 12 with fewer in exon 7. The clinical and biochemical outcomes in some patients are described. We also use the three‐dimensional structure of the porphobilinogen deaminase enzyme to predict the possible molecular and functional consequences of the new Swedish missense and nonsense mutations.

Porphobilinogen deaminase and uroporphyrinogen III synthase: Structure, molecular biology, and mechanism

Porphobilinogen deaminase (hydroxymethylbilane synthase) and uroporphyrinogen III synthase (uroporphyrinogen III cosynthase) catalyze the transformation of four molecules of porphobilinogen, via the 1-hydroxymethylbilane, preuroporphyrinogen, into uroporphyrinogen III. A combination of studies involving protein chemistry, molecular biology, site-directed mutagenesis, and the use of chemically synthesized substrate analogs and inhibitors is helping to unravel the complex mechanisms by which the two enzymes function. The determination of the X-ray structure ofE. coli porphobilinogen deaminase at 1.76 Å resolution has provided the springboard for the design of further experiments to elucidate the precise mechanism for the assembly of both the dipyrromethane cofactor and the tetrapyrrole chain. The human deaminase structure has been modeled from theE. coli structure and has led to a molecular explanation for the disease acute intermittent porphyria. Molecular modeling has also been employed to simulate the spiro-mechanism of uroporphyrinogen III synthase.

A Structural Study of Norovirus 3C Protease Specificity: Binding of a Designed Active Site-Directed Peptide Inhibitor

Noroviruses are the major cause of human epidemic nonbacterial gastroenteritis. Viral replication requires a 3C cysteine protease that cleaves a 200 kDa viral polyprotein into its constituent functional proteins. Here we describe the X-ray structure of the Southampton norovirus 3C protease (SV3CP) bound to an active site-directed peptide inhibitor (MAPI) which has been refined at 1.7 Å resolution. The inhibitor, acetyl-Glu-Phe-Gln-Leu-Gln-X, which is based on the most rapidly cleaved recognition sequence in the 200 kDa polyprotein substrate, reacts covalently through its propenyl ethyl ester group (X) with the active site nucleophile, Cys 139. The structure permits, for the first time, the identification of substrate recognition and binding groups in a noroviral 3C protease and thus provides important new information for the development of antiviral prophylactics.

The three‐dimensional structure of Escherichia coli porphobilinogen deaminase at 1.76‐Å resolution

Porphobilinogen deaminase (PBGD) catalyses the polymerization of four molecules of porphobilinogen to form the 1‐hydroxymethylbilane, preuroporphyrinogen, a key intermediate in the biosynthesis of tetrapyrroles. The three‐dimensional structure of wild‐type PBGD from Escherichia coli has been determined by multiple isomorphous replacement and refined to a crystallographic R‐factor of 0.188 at 1.76 Å resolution. The polypeptide chain of PBGD is folded into three α/β domains. Domains 1 and 2 have a similar overall topology, based on a five‐stranded, mixed β‐sheet. These two domains, which are linked by two hinge segments but otherwise make few direct interactions, form an extensive active site cleft at their interface. Domain 3, an open‐faced, anti‐parallel sheet of three strands, interacts approximately equally with the other two domains. The dipyrromethane cofactor is covalently attached to a cysteine side‐chain borne on a flexible loop of domain 3. The cofactor serves as a primer for the assembly of the tetrapyrrole product and is held within the active site cleft by hydrogen‐bonds and salt‐bridges that are formed between its acetate and propionate side‐groups and the polypeptide chain. The structure of a variant of PBGD, in which the methionines have been replaced with selenomethionines, has also been determined. The cofactor, in the native and functional form of the enzyme, adopts a conformation in which the second pyrrole ring (C2) occupies an internal position in the active site cleft. On oxidation, however, this C2 ring of the cofactor adopts a more external position that may correspond approximately to the site of substrate binding and polypyrrole chain elongation. The side‐chain of Asp84 hydrogen‐bonds the hydrogen atoms of both cofactor pyrrole nitrogens and also potentially the hydrogen atom of the pyrrole nitrogen of the porphobilinogen molecule bound to the proposed substrate binding site. This group has a key catalytic role, possibly in stabilizing the positive charges that develop on the pyrrole nitrogens during the ring‐coupling reactions. Possible mechanisms for the processive elongation of the polypyrrole chain involve: accommodation of the elongating chain within the active site cleft, coupled with shifts in the relative positions of domains 1 and 2 to carry the terminal ring into the appropriate position at the catalytic site; or sequential translocation of the elongating polypyrrole chain, attached to the cofactor on domain 3, through the active site cleft by the progressive movement of domain 3 with respect to domains 1 and 2. Other mechanisms are considered although the amino acid sequence comparisons between PBGDs from all species suggest they share the same three‐dimensional structure and mechanism of activity.

Structure of human porphobilinogen deaminase at 2.8 Å: the molecular basis of acute intermittent porphyria

Mutations in the human PBGD (porphobilinogen deaminase) gene cause the inherited defect AIP (acute intermittent porphyria). In the present study we report the structure of the human uPBGD (ubiquitous PBGD) mutant, R167Q, that has been determined by X-ray crystallography and refined to 2.8 A (1 A=0.1 nm) resolution (Rfactor=0.26, Rfree=0.29). The protein crystallized in space group P2(1)2(1)2 with two molecules in the asymmetric unit (a=81.0 A, b=104.4 A and c=109.7 A). Phases were obtained by molecular replacement using the Escherichia coli PBGD structure as a search model. The human enzyme is composed of three domains each of approx. 110 amino acids and possesses a dipyrromethane cofactor at the active site, which is located between domains 1 and 2. An ordered sulfate ion is hydrogen-bonded to Arg26 and Ser28 at the proposed substrate-binding site in domain 1. An insert of 29 amino acid residues, present only in mammalian PBGD enzymes, has been modelled into domain 3 where it extends helix alpha2(3) and forms a beta-hairpin structure that contributes to a continuous hydrogen-bonding network spanning domains 1 and 3. The structural and functional implications of the R167Q mutation and other mutations that result in AIP are discussed.

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase complexed with two diacid inhibitors

The structures of 5‐aminolaevulinic acid dehydratase complexed with two irreversible inhibitors (4‐oxosebacic acid and 4,7‐dioxosebacic acid) have been solved at high resolution. Both inhibitors bind by forming a Schiff base link with Lys 263 at the active site. Previous inhibitor binding studies have defined the interactions made by only one of the two substrate moieties (P‐side substrate) which bind to the enzyme during catalysis. The structures reported here provide an improved definition of the interactions made by both of the substrate molecules (A‐ and P‐side substrates). The most intriguing result is the novel finding that 4,7‐dioxosebacic acid forms a second Schiff base with the enzyme involving Lys 210. It has been known for many years that P‐side substrate forms a Schiff base (with Lys 263) but until now there has been no evidence that binding of A‐side substrate involves formation of a Schiff base with the enzyme. A catalytic mechanism involving substrate linked to the enzyme through Schiff bases at both the A‐ and P‐sites is proposed.

X-ray structure of a putative reaction intermediate of 5-aminolaevulinic acid dehydratase

The X-ray structure of yeast 5-aminolaevulinic acid dehydratase, in which the catalytic site of the enzyme is complexed with a putative cyclic intermediate composed of both substrate moieties, has been solved at 0.16 nm (1.6 A) resolution. The cyclic intermediate is bound covalently to Lys(263) with the amino group of the aminomethyl side chain ligated to the active-site zinc ion in a position normally occupied by a catalytic hydroxide ion. The cyclic intermediate is catalytically competent, as shown by its turnover in the presence of added substrate to form porphobilinogen. The findings, combined with those of previous studies, are consistent with a catalytic mechanism in which the C-C bond linking both substrates in the intermediate is formed before the C-N bond.

The Biosynthesis of Coproporphyrinogen III

ACKNOWLEDGEMENTS : Thanks are due to Dr. K.-M. Cheung and James Youell for assistance with the Schemes, to Barry Lockyer and William Rees-Blanchard for assistance with the figures and to the editors for their patience. Funding from the BBSRC and Wellcome Trust is gratefully acknowledged.

Continuous coupled assay for 5-aminolevulinate synthase

Publisher Summary This chapter discusses the continuous coupled assay for 5-aminolevulinate synthase. A direct 5-aminolevulinate synthase enzyme assay has been developed on the basis of linking 5-aminolevulinate synthase to pyruvate dehydrogenase. Initial investigations demonstrated that pyruvate dehydrogenase was unaffected by components of the 5-aminolevulinate synthase assay system and vice versa. When 5-aminolevulinate synthase activity was determined using this coupled assay, a linear increase in absorbance was obtained. The rate was dependent on the presence of both succinyl-CoA and glycine and was linear with respect to both time and protein concentration. A Lineweaver–Burk plot of dependence of activity of the Rhodobacter spheroides enzyme on succinyl-CoA gave values for K m and V max of 6 μ M and 138 μ mol/ mg protein/hour, respectively. The K m for glycine was 5 m M . These values agree well with those in the literature. Data obtained using the coupled assay showed excellent agreement with the data obtained using the discontinuous assay.

Dipyrromethane cofactor assembly of porphobilinogen deaminase: Formation of apoenzyme and preparation of holoenzyme

Publisher Summary This chapter outlines the methods for generating the apodeaminase and regenerating the holoenzyme from the apoenzyme. The methods may be adapted for labeling the dipyrromethane cofactor with either radioactive or stable isotopes. Porphobilinogen deaminase catalyzes the formation of preuroporphyrinogen from four molecules of porphobilinogen. Preuroporphyrinogen is a highly unstable 1-hydroxymethylbilane, which acts as the substrate for uroporphyrinogen III synthase to yield uroporphyrinogen III—the common tetrapyrrole precursor for other tetrapyrroles. Porphobilinogen deaminases have been isolated from a number of sources and their properties are well established. In the holoenzyme, the four carboxylic acid groups of the dipyrromethane cofactor interact with highly conserved arginine residues in the Escherichia coli enzyme. Porphobilinogen deaminase lacking the cofactor, termed the “apoenzyme,” may be isolated from genetically engineered bacterial strains in which the ability to synthesize the early precursors, 5-aminolevulinic acid and porphobilinogen, has been disrupted. Preuroporphyrinogen is not only the product of porphobilinogen deaminase and the substrate for uroporphyrinogen III synthase but also the precursor of the dipyrromethane cofactor.