Human porphobilinogen deaminase mutations in the investigation of the mechanism of dipyrromethane cofactor assembly and tetrapyrrole formation

Abstract

Porphobilinogen deaminase mutants that cause acute intermittent porphyria have been investigated as recombinant proteins expressed in Escherichia coli, yielding important insight into the mechanism of dipyrromethane cofactor assembly and tetrapyrrole chain polymerization. A mutation that affects a key catalytic residue, D99G, results in an inactive holo -protein that exists as a complex with two substrate molecules covalently bound to the dipyrromethane cofactor arising from the reaction between the apo -protein and pre-uroporphyrinogen. The R149Q mutant is also devoid of catalytic activity but the mutant protein is unable to assemble the dipyrromethane cofactor from pre-uroporphyrinogen and persists as an unstable, heat-labile apo -protein. The mutant, R173Q, has very low activity and, like R149Q, also exhibits largely as an apo -protein. The inability to reconstitute either R149Q or R173Q with exogenous pre-uroporphyrinogen confirms the importance of these two arginine residues for dipyrromethane cofactor assembly. In contrast, the mutant R167Q exists as a holo -enzyme but the catalytic cycle is severely compromised, leading to the accumulation of stable enzyme-substrate intermediates from the catalytic cycle.

Human hydroxymethylbilane synthase: Molecular dynamics of the pyrrole chain elongation identifies step-specific residues that cause AIP

“…The missing coordinates of the residues 60-78 (part of the active-site loop and adjoining region) were modeled based on the structure of AtHMBS [PDB ID code 4HTG (10)] using Modeler 9v8 (22). The missing residues at the N-terminal (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15)(16)(17)(18)(19)(20) and the C-terminal (residues 358-361) were also modeled.…”

“…Previous studies of the purified Escherichia coli and human enzymes demonstrated the critical importance of a covalently bound dipyrromethane (DPM) cofactor and the occurrence of stable enzyme substrate complexes in the formation of HMB (4,(6)(7)(8). To date, the X-ray crystal structures of E. coli (9), Arabidopsis thaliana (10), Bacillus megaterium (11,12), and human (13,14) HMBS (hHMBS) with their DPM cofactors have been reported.…”

Acute intermittent porphyria – impact of mutations found in the hydroxymethylbilane synthase gene on biochemical and enzymatic protein properties

“…Arg173 is an invariant residue in all known sequences. The mutation of Arg173 to Gln results in an apo form of the enzyme that is incapable of catalysis [26]. The missense mutant p.Arg173Trp in AIP patients has also been found to be inactive [27].…”

Molecular characterisation of acute intermittent porphyria in a cohort of South African patients and kinetic analysis of two expressed mutants

“…The bulky arginine moiety in the mutant may disrupt the docking or covalent binding of the cofactor to the enzyme, while a charge switch in the p.Lys98Glu mutant has no such effect. Several other mutants (Arg149Gln, Arg173Gln38 and Arg116Trp)39 are also reported to result in the expression of apoenzyme. It is suggested that the disrupted interaction of these residues with the cofactor likely prevents successful docking of hydroxymethylbilane, the DPM precursor, in the active site of the apoenzyme.…”