Pyrroloquinoline quinone-dependent dehydrogenases from Ketogulonicigenium vulgare catalyze the direct conversion of L-sorbosone to L-ascorbic acid

Abstract

A novel enzyme, L-sorbosone dehydrogenase 1 (SNDH1), which directly converts L-sorbosone to L-ascorbic acid (L-AA), was isolated from Ketogulonicigenium vulgare DSM 4025 and characterized. This enzyme was a homooligomer of 75-kDa subunits containing pyrroloquinoline quinone (PQQ) and heme c as the prosthetic groups. Two isozymes of SNDH, SNDH2 consisting of 75-kDa and 55-kDa subunits and SNDH3 consisting of 55-kDa subunits, were also purified from the bacterium. All of the SNDHs produced L-AA, as well as 2-keto-L-gulonic acid (2KGA), from L-sorbosone, suggesting that tautomerization of L-sorbosone causes the dual conversion by SNDHs. The sndH gene coding for SNDH1 was isolated and analyzed. The N-terminal four-fifths of the SNDH amino acid sequence exhibited 40% identity to the sequence of a soluble quinoprotein glucose dehydrogenase from Acinetobacter calcoaceticus. The C-terminal one-fifth of the sequence exhibited similarity to a c-type cytochrome with a heme-binding motif. A lysate of Escherichia coli cells expressing sndH exhibited SNDH activity in the presence of PQQ and CaCl2. Gene disruption analysis of K. vulgare indicated that all of the SNDH proteins are encoded by the sndH gene. The 55-kDa subunit was derived from the 75-kDa subunit, as indicated by cleavage of the C-terminal domain in the bacterial cells.

FIG. 1.

PAGE analysis of SNDHs. (A) Activity staining of the l-sorbosone-dehydrogenating enzymes of K. vulgare DSM 4025 cell extract on a native 10% polyacrylamide gel. The locations of SNDH, SSDH, and ALDH proteins are indicated on the left. (B) SDS-PAGE (12.5% polyacrylamide gel) analysis of purified SNDHs. Lane MW contained a mixture of molecular mass markers. Purified SNDH1 (1.0 μg), SNDH2 (1.3 μg), and SNDH3 (0.75 μg) were loaded into the lanes. The proteins were stained with CBB.

FIG. 2.

(A) Reconstruction of holo-SNDH from apo-SNDH prepared by electroelution from a native polyacrylamide gel. In each lane, 0.75 μg of the protein was used. Lanes 1 and 2, activity staining of l-sorbosone-dehydrogenating enzymes; lanes 3 and 4, protein staining with CBB. Lanes 1 and 3 contained apo-SNDH, and lanes 2 and 4 contained SNDH treated with 1 μM PQQ and 1 mM CaCl₂. The solid arrowhead indicates the position of the apoenzyme. The open arrowhead indicates the position of the holoenzyme in which PQQ binds to the apoenzyme. (B) Reduced-minus-oxidized difference spectra of the purified SNDHs.

FIG. 3.

Nucleotide and deduced amino acid sequences for the sndH region in K. vulgare. Thin lines indicate putative promoter sequences (TAGACC and TAACCT). The putative Shine-Dalgarno (SD) sequence is indicated by a boldface line. Inverted repeat sequences are indicated by pairs of inverted arrows (IR 1, IR 2, and IR 3). Stop codons are indicated by asterisks. For sndH, the signal sequence is indicated by a dashed line, the N terminus of the mature SNDH polypeptide is indicated by an arrowhead, and the heme-binding motif is enclosed in a box. For deaD, the DEAD motif is enclosed in a box.

FIG. 4.

Gene organization of sndH region in K. vulgare. Km, insertion site of the kanamycin resistance gene in GOMTR1SN::Km; Heme, heme c binding motif; solid arrows, coding regions of 55-kDa and 75-kDa subunits. Phe³⁰⁸, Thr⁴²⁹, and Gly⁵³⁵ indicate the C termini of the truncated SNDH molecules encoded in deletion plasmids.

FIG. 5.

Expression of intact and truncated sndH genes in E. coli (A) and K. vulgare (B). (A) E. coli cells harboring pUCSN series plasmids: activity staining (upper gel) and Western blot analysis (lower gel) of CFE proteins. For the upper gel, CFE proteins (15 μg) were incubated with PQQ and CaCl₂ for 10 min at 25°C and subjected to native PAGE (10% polyacrylamide gel), followed by activity staining using l-sorbosone. The locations of SNDH, SSDH, and ALDH proteins are indicated on the left. The open arrowhead indicates the position of the fourth SNDH band. For the lower gel, CFE proteins (5 μg) were subjected to SDS-PAGE (12.5% polyacrylamide gel) and blotted onto a polyvinylidene difluoride membrane. The protein bands were detected using rabbit polyclonal anti-SNDH antiserum. The locations of 75-kDa and 55-kDa subunits are indicated on the left. Lane 1, K. vulgare DSM 4025; lanes 2 to 6, E. coli JM109 harboring pUCSN2004 (lane 2), pUCSN2003 (lane 3), pUCSN2002 (lane 4), pUCSN2001 (lane 5), and pUC18 (lane 6). (B) K. vulgare sndH::Km cells harboring pVSN series plasmids. CFE proteins (16 μg) were subjected to native PAGE (10% polyacrylamide gel), followed by activity staining using l-sorbosone. Lane 1, K. vulgare GOMTR1; lane 2, GOMTR1SN::Km; lanes 3 to 6, GOMTR1SN::Km harboring pVSN117 (lane 3), pVSN106 (lane 4), pVSN114 (lane 5), and pVK100 (lane 6).

FIG. 6.

Proposed metabolic pathways from d-sorbitol to l-AA and 2KGA in K. vulgare DSM 4025. Reaction 1 is catalyzed by SSDH (3); reaction 2 is catalyzed by ALDH (15); and reactions 3a and 3b are catalyzed by SNDHs.