Gene expression and characterization of 2-keto-3-deoxy-gluconate kinase, a key enzyme in the modified Entner-Doudoroff pathway of Serratia marcescens KCTC 2172

ABSTRACT

We cloned 2-keto-3-deoxy-gluconate kinase (KDGK), which catalyzes the phosphorylation of 2-keto-3-deoxygluconate (KDG) to 2-keto-3-deoxy-6-phophogluconate (KDPG) from Serratia marcescens KCTC 2172. The nucleotide sequence revealed a single open reading frame containing 1,208 bp and encoding for 309 amino acids, with a molecular weight of 33,993 Da. The enzyme was purified via GST affinity chromatography. The putative KdgT binding site was detected upstream of the initial codon. The KDG kinase utilized 2-ketogluconate (KG) and KDG as substrates. The optimal temperature and pH for KDGK activity were 50ºC and 8.0, respectively.