Identification of lactaldehyde dehydrogenase in Methanocaldococcus jannaschii and its involvement in production of lactate for F420 biosynthesis

Abstract

One of the early steps in the biosynthesis of coenzyme F(420) in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NAD(+)-dependent oxidation of l-lactaldehyde by the MJ1411 gene product. The lactaldehyde, in turn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-1-phosphate by fuculose-1-phosphate aldolase, the MJ1418 gene product.

FIG. 1.

Sequence alignment of M. jannaschii lactaldehyde dehydrogenase with lactaldehyde dehydrogenases from E. coli (encoded by aldB, NP_418045) and Salmonella enterica serovar Typhimurium (encoded by aldB, NP_462580) as well as T. tenax glyceraldehyde-3-phosphate dehydrogenase (T44939). Identical residues are shown in boldface, and similar residues are highlighted in gray. Conserved catalytic cysteine and glutamate residues are indicated with an asterisk. Residues proposed to be involved in glyceraldehyde-3-phosphate dehydrogenase substrate binding are highlighted in black.

FIG. 2.

Fuculose-1-phosphate aldolase reaction products. Shown is an analysis of the reduced and dephosphorylated reaction products resulting from the condensation of d- and l-lactaldehyde with DHAP. The four peaks observed were generated by reduction of the two products resulting from the condensation of DHAP with l- and d-lactaldehyde, respectively. (A) GC-MS fragmentation pattern of unlabeled TMS₅ products. (B) GC-MS fragmentation pattern of products reduced with borodeuteride. (C) GC-MS fragmentation pattern of product formed from lactaldehyde labeled with deuterium on C-2. Although the fragmentation pattern is drawn for the fucitol (TMS)₅ derivative, an identical fragmentation pattern was seen in the corresponding peak for each stereoisomer.

FIG. 3.

Pathway for lactate formation in M. jannaschii.