Enzyme and Microbial Technology 32 (2003) 27–34 Inhibition of 5-aminolevulinic acid dehydratase in recombinant Escherichia coli using d-glucose Dae-Hee Lee, Woo-Jin Jun, Kyung-Mi Kim, Dong-Hoon Shin, Hong-Yon Cho, Bum-Shik Hong ∗ Graduate School of Biotechnology, Korea University, #1, 5-ka, Anam-Dong, Sungbuk-ku, Seoul 136-701, South Korea Received 9 April 2002; received in revised form 21 August 2002; accepted 26 August 2002 Abstract For the overproduction of 5-aminolevulinic acid (ALA) from recombinant Escherichia coli, the inhibition of ALA dehydratase on both small scale by using an eppendorf tube, and on a large scale by using a fermenter, the in vitro glycation and the inactivation of enzymes on the ALA dehydratase under several experimental conditions were investigated. The presence of 0.5–10mM of d-glucose caused a concentration-dependent inhibition of recombinant E. coli ALA dehydratase activity. The ALA dehydratase levels were dependent on the pH of the medium, with the maximal activities occurring at 8.0. The inhibition constant, K i , of intracellular ALA dehydratase by d-glucose and levulinic acid (LA) were 1.02 and 0.32 mM, respectively. The addition of 10 mM of d-glucose drastically inhibited the ALA dehydratase activity (85% inhibition), in turn, the highest level of extracellular ALA production (3.8g/l) was achieved. Based upon those results, we concluded that d-glucose decreased the ALA dehydratase activity both by the competitive inhibition with substrate and by the inactivation of enzyme protein, and that the inactivation of ALA dehydratase by d-glucose may require glycation metabolism of d-glucose at least in part. © 2002 Elsevier Science Inc. All rights reserved. Keywords: Aminolevulinic acid; Aminolevulinic acid dehydratase; Recombinant Escherichia coli; d-Glucose 1. Introduction 5-Aminolevulinic acid (ALA) is a precursor for tetrapyrroles in all living systems [1,2]. Many researchers have investigated for a better alternative production of ALA. Choi et al. [3] reported the overproduction of ALA by recombinant Escherichia coli containing Bradyrhizobium japonicum hemA gene. They examined both enzymatic and physiological factors affecting the expression of Bradyri- zobium japonicum hemA in E. coli (BL21DE3) and the production of ALA. Using this “molecular physiology” ap- proach, they produced a higher amount of ALA than had been reported before. For the high production of ALA, the inhibition of ALA dehydratase is essential. The ALA dehydratase, also known as porphobilinogen (PBG) synthase (EC 4.2.1.24), is a met- alloprotein enzyme that catalyses the asymmetric formation of one molecule of porphobilinogen from two molecules of ALA (Fig. 1). This reaction involves the aldol condensa- tion between two o-ALA molecules, the formation of a C–N ∗ Corresponding author. Tel.: +82-2-3290-3926; fax: +82-2-3290-3956. E-mail address: bumshik@korea.ac.kr (B.-S. Hong). bond, and the elimination of water molecules to produce por- phobilinogen, forporphyrins or other tetrapyrrole-like com- pounds [4]. With enzyme-catalyzed aldol condensation and Schiff base formation, the reaction promoted by ALA de- hydratase lead to the production of an enzyme–substrate Schiff base intermediate [5]. A single molecule of levulinic acid (LA) binds to a mainly hydrophobic pocket in each monomer where it is covalently attached via a Schiff base to an active site lysine residue. The ALA dehydratase can be inhibited by either substrate—or product-analogues. As utilizing the most widely used substrate analogue, LA, the high amounts of ALA could be produced in various organ- isms [6]. In the present work, we selected d-glucose among various inhibitors, because d-glucose is non-toxic and eco- nomic compared to other inhibitors. Glycation is the first of a series of reactions known as “Maillard browning” [7]. The aldehyde group of d-glucose is known to react with amino groups of proteins, result- ing in a Schiff base conjugate formation, which subse- quently undergoes an Amadori rearrangement leading to a stable ketoamine adduct. This process can promote the cross-linking of proteins through ε-NH 2 groups of lysine or hydroxylysine. 0141-0229/02/$ – see front matter © 2002 Elsevier Science Inc. All rights reserved. PII:S0141-0229(02)00241-7