Cloning and expression of a gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase from Erwinia cypripedii ATCC 29267 in Escherichia coli

Abstract

We have cloned the gene cluster encoding three subunits of membrane-bound gluconate dehydrogenase (GADH) from Erwinia cypripedii ATCC 29267 in Escherichia coli by performing a direct-expression assay. The positive clone converted D-gluconate to 2-keto-D-gluconate (2KDG) in the culture medium. Nucleotide sequence analysis of the GADH clone revealed that the cloned fragment contained the complete structural genes for a 68-kDa dehydrogenase subunit, a 47-kDa cytochrome c subunit, and a 24-kDa subunit of unknown function and that the genes were clustered with the same transcriptional polarity. Comparison of the deduced amino acid sequences and the NH 2 -terminal sequences determined for the purified protein indicated that the dehydrogenase, cytochrome c, and 24-kDa subunits contained typical signal peptides of 22, 19, and 42 amino acids, respectively. The molecular masses of the processed subunits deduced from the nucleotide sequences (65, 45, and 20 kDa) coincided well with the molecular masses of subunits estimated by sodium dodecyl sulfatepolyacrylamide gel electrophoresis. In E. cypripedii and recombinant E. coli, the GADH was constitutively formed and the activity of GADH was enhanced more than twofold by addition of D-gluconate to the medium. The conversion of D-glucose to D-gluconate, 2-keto-D-gluconate (2KDG), and 2,5-diketo-D-gluconate (25DKG) in several species of the genera Acetobacter, Gluconobacter, and Erwinia is mediated by membrane-bound dehydrogenases linked to the cytochrome chains located in the cytoplasmic membrane of the bacterium (1, 31). Membrane-bound gluconate dehydrogenases (GADHs), which catalyze the production of 2KDG from D-gluconate, have been purified and characterized from Pseudomonas aeruginosa (16,19), P. fluorescens (17), Klebsiella pneumoniae (17), Serratia marcescens (17), and Gluconobacter dioxyacetonicus (28). The subunit structures and enzymatic properties of these GADHs were found to be very similar (17). These GADHs consisted of three subunits; namely, flavoprotein, cytochrome c, and a third subunit whose function is unknown. Despite these findings, no further work on gene structures has been reported.In the present work, we describe the direct-expression cloning of the gene cluster encoding three subunits of membranebound GADH from Erwinia cypripedii ATCC 29267. E. coli K-12 derivatives are capable of synthesizing the apo-glucose dehydrogenase (apo-GDH) but not the cofactor, pyrroloquinoline quinone (PQQ), which is essential for the formation of the holoenzyme (3, 5). When PQQ is present in the medium, the holoenzyme is reconstituted and then E. coli is capable of oxidizing glucose to gluconate (7). It has also been reported that the expression of PQQ synthase genes in E. coli resulted in GDH activity in the absence of exogenous PQQ (13). Therefore, we tried to convert D-glucose to 2KDG via D-gluconate by using recombinant E. coli harboring the cloned GADH gene in the presence of PQQ. MATERIALS AND METHODSBacterial strains, media, and culture conditions. E. cypripedii ATCC ...

The PA4204 gene encodes a periplasmic gluconolactonase (PpgL) which is important for fitness of Pseudomonas aeruginosa

“…The PA4204 enzyme is probably a periplasmic gluconolactonase active against either gluconolactone or a lactone compound (2-dehydro-D-glucono-1,5-lactone) that originates spontaneously from 2-ketogluconate, which itself is the product of the oxidation of gluconate by the membrane-bound gluconate dehydrogenase encoded by gad (PA2265). In Pectobacterium (Erwinia) cypripedii, the gluconate dehydrogenase is a three-component enzyme comprising a small subunit, the Gad flavoprotein and a c-type cytochrome (Yum et al, 1997). In P. aeruginosa, the same genes (PA2264-PA2266) are found in the same order as in Pectobacterium (Erwinia) cypripedii (http://www.pseudomonas.com).…”

Molecular Properties of Membrane-Bound FAD-ContainingD-Sorbitol Dehydrogenase from ThermotolerantGluconobacter frateuriiIsolated from Thailand

“…From bacteria other than acetic acid bacteria, membrane-bound GADH and 2KGDH from E. cypripedii 32) and P. agglomerans or P. citrea 33) respectively have been reported to contain similar cytochrome c subunits. These cytochrome c subunits show high identity to each other in amino acid sequence (Fig.…”

Genetic and Biochemical Characterization of the Pathway in Pantoea citrea Leading to Pink Disease of Pineapple

“…Depending on the start codon used, orfC would encode for a protein of 396 or 357 aa. This putative OrfC protein is 60% similar to the alcohol dehydrogenase cytochrome c subunit of A. polyoxogenes (38) and the cytochrome c precursor of the membrane-bound gluconate dehydrogenase from E. cypripedii (43). Three cytochrome c family heme-binding sites were found at nt 3018 to 3032 (CQACH), 3462 to 3476 (CDTCH) and 3888 to 3902 (CASCH).…”

“…The putative gene product encoded by orfB shares 34% similarity with a gluconate dehydrogenase subunit precursor from Erwinia cypripedii (data not shown) (43). Four potential start codons (ATG) for orfB are located at positions 1225, 1228, 1231, and 1408 (Fig.…”

“…Furthermore, a number of Acetobacter, Erwinia, and Gluconobacter species are able to oxidize 2-KDG to 2,5-DKG by membrane bound 2-ketogluconate dehydrogenases (5,43). We therefore decided to test these different compounds for the ability to be used as a substrate for the dark coloration characteristic of pink disease.…”