The ald gene, encoding a coenzyme A-acylating aldehyde dehydrogenase, distinguishes Clostridium beijerinckii and two other solvent-producing clostridia from Clostridium acetobutylicum

Abstract

The coenzyme A (CoA)-acylating aldehyde dehydrogenase (ALDH) catalyzes a key reaction in the acetone- and butanol (solvent)-producing clostridia. It reduces acetyl-CoA and butyryl-CoA to the corresponding aldehydes, which are then reduced by alcohol dehydrogenase (ADH) to form ethanol and 1-butanol. The ALDH of Clostridium beijerinckii NRRL B593 was purified. It had no ADH activity, was NAD(H) specific, and was more active with butyraldehyde than with acetaldehyde. The N-terminal amino acid sequence of the purified ALDH was determined. The open reading frame preceding the ctfA gene (encoding a subunit of the solvent-forming CoA transferase) of C. beijerinckii NRRL B593 was identified as the structural gene (ald) for the ALDH. The ald gene encodes a polypeptide of 468 amino acid residues with a calculated M(r) of 51, 353. The position of the ald gene in C. beijerinckii NRRL B593 corresponded to that of the aad/adhE gene (encoding an aldehyde-alcohol dehydrogenase) of Clostridium acetobutylicum ATCC 824 and DSM 792. In Southern analyses, a probe derived from the C. acetobutylicum aad/adhE gene did not hybridize to restriction fragments of the genomic DNAs of C. beijerinckii and two other species of solvent-producing clostridia. In contrast, a probe derived from the C. beijerinckii ald gene hybridized to restriction fragments of the genomic DNA of three solvent-producing species but not to those of C. acetobutylicum, indicating a key difference among the solvent-producing clostridia. The amino acid sequence of the ALDH of C. beijerinckii NRRL B593 was most similar (41% identity) to those of the eutE gene products (CoA-acylating ALDHs) of Salmonella typhimurium and Escherichia coli, whereas it was about 26% identical to the ALDH domain of the aldehyde-alcohol dehydrogenases of C. acetobutylicum, E. coli, Lactococcus lactis, and amitochondriate protozoa. The predicted secondary structure of the C. beijerinckii ALDH suggests the presence of an atypical Rossmann fold for NAD(+) binding. A comparison of the proposed catalytic pockets of the CoA-dependent and CoA-independent ALDHs identified 6 amino acids that may contribute to interaction with CoA.

FIG. 1

Southern hybridization analysis of four species of solvent-producing clostridia for the presence of DNA sequences related to the ald gene of C. beijerinckii NRRL B593. Lanes B and H contained, respectively, BglII- and HindIII-digested genomic DNA of the following strains: C. acetobutylicum ATCC 824 (type strain) and NRRL B528; C. beijerinckii VPI 5481 (type strain), NCIMB 8052, NCIMB 6444, and NRRL B593; two strains (NCP 262 and NRRL B643) belonging to an unnamed species; and “C. saccharoperbutylacetonicum” N1-4. The border lanes contained 1-kb ladders.

FIG. 2

Southern hybridization analysis of four species of solvent-producing clostridia for the presence of DNA sequences related to the aad/adhE gene of C. acetobutylicum. Strains tested were those listed in the legend to Fig. 1. Lanes B and H contained, respectively, BglII- and HindIII-digested genomic DNA of each strain. The border lanes contained 1-kb ladders.

FIG. 3

Southern hybridization analysis of strains of C. beijerinckii for the presence of DNA sequences related to the ald gene of C. beijerinckii NRRL B593. Lanes B and H contained, respectively, BglII- and HindIII-digested genomic DNA of the indicated strains of C. beijerinckii. The border lanes contained 1-kb ladders.

FIG. 4

Alignment of the amino acid sequences of three CoA-dependent ALDHs and the N-terminal region of an ALDH-related protein. Markers above the sequences indicate the positions used in the alignment of the C. beijerinckii NRRL B593 ALDH. The sources and abbreviations are as follows: Oct Crys, Ω-crystallin of the giant octopus (41); Cb ALDH, ALDH of C. beijerinckii NRRL B593 (accession no. AF132754); St EutE, the eutE-encoded ALDH of S. typhimurium (AAA80209); Ca Aad, the ALDH domain encoded by the aad gene of C. acetobutylicum ATCC 824 (25). Amino acid residues that are shared by the C. beijerinckii NRRL B593 ALDH and at least one of the other sequences are highlighted. Positions of amino acids that line the proposed catalytic pocket of the rat ALDH3 are indicated by an asterisk or a caret (the latter indicates a residue that is conserved in nine CoA-dependent ALDHs but not in CoA-independent ALDHs at these positions). An underlined number in a sequence indicates the number of amino acids omitted at that position to save space. Dashes in the sequence indicate the gaps that were introduced to refine the alignment. Boldfaced letters above the sequences indicate amino acid residues conserved in previously aligned ALDHs with demonstrated dehydrogenase activity (28).

FIG. 5

Alignment of the region of the amino acid sequence that forms the Rossmann fold in the class 3 ALDH of rat (Rat ALDH3) with the corresponding region of the ALDH of C. beijerinckii NRRL B593 (Cb ALDH). Positions of amino acids that are identical in the two sequences are linked by vertical lines; those occupied by similar amino acids are linked by colons. The predicted secondary structure of the C. beijerinckii NRRL B593 ALDH is compared with the X-ray crystal structure of the rat ALDH3 (24). The α-helices and β-sheets are marked, respectively, by circles and carets either above or below the amino acid sequence. The secondary structures in the NAD-binding domains of the two ALDHs that are apparently conserved are highlighted below the C. beijerinckii ALDH sequence. Amino acid residues that line the proposed catalytic pocket of the rat ALDH3 are marked by asterisks above the sequence. Dashes in the sequence indicate the gaps that were introduced to refine alignment.