Characterization of L-Lysine 6-Aminotransferase and Its Structural Gene from Flavobacterium lutescens IFO3084

Abstract

L-Lysine 6-aminotransferase (LAT) is an enzyme involved in L-lysine catabolism in a wide range of living organisms. LAT from Flavobacterium lutescens IFO3084 was purified, and its structural gene (lat) was cloned, sequenced and expressed in Escherichia coli. Native PAGE analysis of purified LAT gave a single band corresponding to a molecular weight of about 110,000. lat encoded a protein of 493 amino acids with a deduced molecular weight of 53,200, which is very close to that of purified LAT determined on SDS-PAGE. Expression of lat in E. coli revealed that lat encodes a single subunit protein leading to LAT activity. These data suggested that LAT from F. lutescens IFO3084, like most other aminotransferases, is derived from a single ORF and is active as a homodimer.

Biotransformation ofL-Lysine toL-Pipecolic Acid Catalyzed byL-Lysine 6-Aminotransferase and Pyrroline-5-carboxylate Reductase

“…Recombinant LAT and recombinant P5C reductase were expressed and puriˆed using E. coli TOP10 W pTrcLAT and E. coli TOP10 W pTrcP5CR, respectively, by the method described previously. 10) Cooperative enzymatic transformation of L-Lys to L-PA in vitro. Reaction mixture contained 0.2 mmol of sodium phosphate buŠer (pH 7.2), 1 mmol of Llysine HCl, 10 mmol of 2-ketoglutarate, 37.5 pmol of pyridoxsal phosphate, 2 mmol of NADPH, 1 mg of LAT, and 5 mg of P5C reductase in aˆnal volume of 1 ml.…”

“…2A). Since LAT catalyzes the conversion of L-Lys to P6C, 6,10) this result implies that there is a gene encoding another`P 6C reductase'' also in the genome of E. coli JM109.…”

“…10,11) Our next attention was focused on the``P6C reductase'' that catalyzes the reduction of P6C to L-PA to produce L-PA from LLys. We found that``P6C reductase'' exists in Escherichia coli.…”

Investigation of the biosynthesis of the pipecolate moiety of neuroprotective polyketide meridamycin

“…The e-deamination of L-lysine to form P6C can be catalyzed by a lysine-6-aminotransferase (for example, in the b-lactam antibiotic producing actinomycetes Streptomyces clavuligerus 19 and Nocardia lactamdurans, 20 or gram-negative bacteria Flavobacterium lutescence 21 ) or by an L-lysine-6-dehydrogenase as in Agrobacterium tumefaciens. 22 The enzyme dedicated to the second step in this pathway, the reduction of P6C to form pipecolate, has not been identified in bacteria.…”

Increase in the Rate ofL-Pipecolic Acid Production Usinglat-ExpressingEscherichia colibylysPandyeiEAmplification

“…We have shown that L-lysine (L-Lys) is converted to L-PA via D 1 -piperideine-6-carboxylate (P6C) using L-lysine 6-aminotransferase (LAT, EC 2.6.1.36) encoded by lat from Flavobacterium lutescens IFO3084 7) and pyrroline-5-carboxylate reductase (P5C reductase, EC 1.5.1.2) encoded by proC from Escherichia coli (Fig. 1).…”