Site-directed mutagenesis and high-resolution NMR spectroscopy of the active site of porphobilinogen deaminase

Abstract

The active site of porphobilinogen (PBG)1 deaminase (EC 4.3.1.8) from Escherichia coli has been found to contain an unusual dipyrromethane derived from four molecules of 5-aminolevulinic acid (ALA) covalently linked to Cys-224, one of the two cysteine residues conserved in E. coli and human deaminase. By use of a hemA- strain of E. coli the enzyme was enriched from [5-13C]ALA and examined by 1H-detected multiple quantum coherence spectroscopy, which revealed all of the salient features of a dipyrromethane composed of two PBG units linked head to tail and terminating in a CH2-S bond to a cysteine residue. Site-specific mutagenesis of Cys-99 and Cys-242, respectively, has shown that substitution of Ser for Cys-99 does not affect the enzymatic activity, whereas substitution of Ser for Cys-242 removes essentially all of the catalytic activity as measured by the conversion of the substrate PBG to uro'gen I. The NMR spectrum of the covalent complex of deaminase with the suicide inhibitor 2-bromo-[2,11-13C2]PBG reveals that the aninomethyl terminus of the inhibitor reacts with the enzyme's cofactor at the alpha-free pyrrole. NMR spectroscopy of the ES2 complex confirmed a PBG-derived head-to-tail dipyrromethane attached to the alpha-free pyrrole position of the enzyme. A mechanistic rationale for deaminase is presented.

Insights into the mechanism of pyrrole polymerization catalysed by porphobilinogen deaminase: high-resolution X-ray studies of theArabidopsis thalianaenzyme

“…A model built in this way may mimic one of the conformational states of the ES 2 intermediate which is formed during elongation of the polypyrrole chain. Interestingly, this intermediate has been shown to be more stable than either of the other isolable intermediates ES 1 or ES 3 (Scott et al, 1988;Warren & Jordan, 1988), suggesting that the enzyme active site can accommodate a covalently bound linear tetrapyrrole particularly favourably. This is expected since the cofactorassembly process involves the binding of the tetrapyrrole preuroporphyrinogen (S 4 ) to the apoenzyme (Awan et al, 1997).…”

“…NMR, isotopic labelling and mass-spectrometric studies of the Escherichia coli PBGD enzyme showed that it possesses a dipyrromethane cofactor (Fig. 2) which is covalently bound to the enzyme by a thioether linkage involving an invariant cysteine residue (Cys242 in E. coli numbering; Jordan & Warren, 1987;Warren & Jordan, 1988;Scott et al, 1988). Whilst the apoenzyme possesses no catalytic activity, incubation with PBG for a period of several hours at pH 8.0 generates active holoenzyme (Scott et al, 1989).…”

A Kinetic Analysis of the Reaction Catalysed by (Hydroxymethyl)bilane Synthase

“…A representative example is the replacement of Cys-242 by Ser ([Ser242]HMBS). The resulting mutant enzyme lacks the anchor to which the dipyrrin cofactor has to be attached and indeed shows no activity [31]. Other deleterious replacements include the one of arginine residues at positions 11, 131, 132, and 155 by leucine or histidine, but in these cases, the mechanistic reasons are not well understood yet.…”

Purification and properties of porphobilinogen deaminase from Arabidopsis thaliana.

“…Deaminases are unique in that they all utilize a dipyrromethane cofactor as a primer to which the four substrate molecules are covalently attached (Jordan and Warren, 1987;Hart et al, 1987;Warren and Jordan, 1988). The dipyrromethane cofactor is covalently attached to Cys-242 in the Escherichia coli deaminase sequence (Jordan et al, 1988a;Miller et al, 1988;Scott et al, 1988).…”