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 Preparation and characterization of mutants in which (a) Lys-55, (b) Lys-59 and (c) both Lys-55 and Lys-59 have been replaced by glutamine

Abstract

A new construct carrying the hemC gene was transformed into Escherichia coli, resulting in approx. 1000-fold overexpression 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 kaCpp IKapp . 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.

Structure and Expression of Chloroplast-Localized Porphobilinogen Deaminase from Pea (Pisum sativum L.) Isolated by Redundant Polymerase Chain Reaction

“…Supporting this notion is the observation that residues essential for catalysis identified by site-directed mutagenesis of E. coli PBG deaminase (Hadener et al, 1990;Jordan and Woodcock, 1991;Lander et al, 1991) are conserved in the pea enzyme. These include pea residues that correspond to E. coli Arg's at positions 11,131,132,149,155,176, and 232, which are involved in substrate binding, and CysZ4', to which the dipyrromethane cofactor is bound (solid triangles in Fig.…”

“…A number of conserved residues have been shown by chemical modification studies or site-directed mutagenesis of the Escherichia coli enzyme to be important for catalytic activity. Among these conserved residues is CysZ4', which binds the dipyrromethane cofactor Miller et al, 1988), severa1 Arg's involved in substrate binding (Jordan and Woodcock, 1991;Lander et al, 1991), and putative active site Lys's (Hadener et al, 1990). The recent determination of tbe three-dimensional structure of the E. coli enzyme at 1.9 A resolution (Louie et al, 1992) will greatly facilitate the study of structure-function relationships in this enzyme.…”

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

“…The purification of wild-type and [SeMetIHMBS was essentially carried out following an established procedure for wild-type HMBS [17]. In brief, thawed cells were resuspended in 0.1 M phosphate (pH 8.0) and sonicated.…”

“…Both E. coli PO1423 and PO1562 were transformed with pPA410 [17] and stocks of the resultant strains were kept over liquid nitrogen.…”

“…A suitable system for the over-expression of E. coli HMBS was already available [17]. It is based on the plasmid pPA410 carrying the tuc promoter upstream of a 1.7-kb BamHI-SaZI fragment of pLC41-4, containing the hemC gene (coding for HMBS) [18, 191. Here we describe the construction of a new Met auxotroph of E. coli (carrying a metEallele), suitable as host for the plasmid pPA410, the expression, in the presence of SeMet, of [SeMetlHMBS after transformation of the E. coli metE mutant strain (P01562) with the latter plasmid, the purification and crystallisation of the SeMet-containing enzyme, the analysis of its kinetic properties and amino acid composition, and its characterization by electrospray mass spectroscopy and X-ray diffraction analysis.…”