NADP+-Preferring D-Lactate Dehydrogenase from Sporolactobacillus inulinus

Abstract

Hydroxy acid dehydrogenases, including l- and d-lactate dehydrogenases (L-LDH and D-LDH), are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids and extensively used in a wide range of biotechnological applications. A common feature of LDHs is their high specificity for NAD(+) as a cofactor. An LDH that could effectively use NADPH as a coenzyme could be an alternative enzymatic system for regeneration of the oxidized, phosphorylated cofactor. In this study, a d-lactate dehydrogenase from a Sporolactobacillus inulinus strain was found to use both NADH and NADPH with high efficiencies and with a preference for NADPH as its coenzyme, which is different from the coenzyme utilization of all previously reported LDHs. The biochemical properties of the D-LDH enzyme were determined by X-ray crystal structural characterization and in vivo and in vitro enzymatic activity analyses. The residue Asn(174) was demonstrated to be critical for NADPH utilization. Characterization of the biochemical properties of this enzyme will contribute to understanding of the catalytic mechanism and provide referential information for shifting the coenzyme utilization specificity of 2-hydroxyacid dehydrogenases.

FIG 1

HPLC analyses of products of pyruvate reduction by purified DLDH744 with NADPH or NADH as cofactors. (a) Reaction with NADPH as coenzyme; (b) reaction with NADH as coenzyme; (c) control without a coenzyme added; (d) l-lactic acid standard; (e) d-lactic acid standard.

FIG 2

Overall crystal structure of DLDH744. (a) Crystal structure of one subunit in complex with cofactor NAD⁺. The secondary structures are colored in blue for α helix and green for β strand; NAD⁺ is colored in magenta and shown in sticks. (b) Dimeric structure of DLDH744. The other subunit in the dimer is represented by surface and colored in orange.

FIG 3

Structural analysis for identification of cofactor NAD⁺-binding site of DLDH744. Key amino acids necessary for cofactor NAD⁺ binding are shown in sticks, and the hydrogen bonds are shown as dashed lines.

FIG 4

Sequence alignment of DLDH744 with homologous enzymes. The secondary structure of D-LDH from L. bulgaricus (PDB ID 1J4A) is presented (top). The indicated numbers of the residues refer to the D-LDH from L. bulgaricus. Helices are marked with spirals, beta strands with arrows, and turns with the letter T. Identical residues and conserved substitutions are shaded in red and boxed in by blue rectangles, respectively. The cofactor-binding sites are identified by a black square. The nucleotide-binding signature domain GXGXXG(17X)D is identified by black triangles. Asn¹⁷⁴ is a unique residue in DLDH744 that differs from residues in the same position in the other d-2-hydroxyacid dehydrogenases. L. pentosus, Lactobacillus pentosus; S. aureus, Staphylococcus aureus.

FIG 5

Comparison between the structures of DLDH744 modeled with NADP⁺ and D-LDH from Aquifex aeolicus in complex with NAD⁺. (a) Crystal structure of DLDH744 modeled with NADP⁺ in the cofactor binding site. The secondary structures are colored in blue for α-helix and green for β-strand; NAD⁺ is colored in magenta and shown in sticks. Asn¹⁷⁴ and other amino acids near the adenine ribose are shown in sticks. (b) The crystal structure of D-LDH from A. aeolicus bound with NAD⁺ was shown in same color scheme as DLDH744. Tyr¹⁷⁰ and corresponding amino acids in D-LDH from A. aeolicus are shown in sticks.

FIG 6

d-Lactate dehydrogenase activity with the cofactors NADH and NADPH after site-directed mutagenesis. Lactate dehydrogenase activity of DLDH744 mutant enzymes with NADH (a) or with NADPH (b) as coenzyme. The error bars in the figure indicate the standard deviations from three parallel replicates.