NADP ⁺ -Preferring d -Lactate Dehydrogenase from Sporolactobacillus inulinus

Abstract

e 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.L actic acid, existing as D-and L-isomers, is an important organic acid that can be widely used in food, cosmetic, chemical, and many other industries (1, 2). The most important application of lactic acid is the production of biodegradable polymer poly(lactic acid) (3). Since the thermoresistance of pure poly(L-lactic acid) is rather low, development of a stereocomplex of poly(L-lactic acid) and poly(D-lactic acid) with a melting point as high as that of petroleum-based polymers is considered an advance in poly(lactic acid) modification (4, 5).L-Lactate dehydrogenase (L-LDH) and D-lactate dehydrogenase (D-LDH) are responsible for the synthesis of L-and D-lactic acids, respectively (2, 6). Given the potential use of chiral hydroxyl acids as valuable synthons and precursors for chiral products in the biotechnological industry (7), it is of significant interest to gain in-depth insights into the substrate specificities, catalytic kinetics, and molecular mechanisms of 2-hydroxyacid dehydrogenase enzymes. The L-isomer-specific 2-hydroxyacid dehydrogenase is a cytoplasmic enzyme present in essentially all major organ systems. This enzyme has been extensively characterized and used as an indicator of pathological conditions (8). However, the D-isomer-specific 2-hydroxyacid dehydrogenase has gained much less attention. Sequence alignments have shown that D-LDH and L-LDH display significant differences in their amino acid sequences and belong to two distinct families: the D-and L-2-hydroxyacid dehydrogenases, respectively (9).Hydroxy acid dehydrogenases are responsible for the stereospecific conversion of 2-keto acids to 2-hydroxyacids, having wide biotechnological applications, like synthesis of antibiotics, flavor development in dairy products, and production of valuable synthons (10). Due to their highly specific a...

Relative catalytic efficiencies and transcript levels of three d‐ and two l‐lactate dehydrogenases for optically pure d‐lactate production in Sporolactobacillus inulinus

“…Zhu et al. found a molecular mass of approximately 37 kDa for DLDH744 enzyme, as determined by SDS‐PAGE, whereas native PAGE analysis indicated a molecular weight of approximately 70 kDa (Zhu et al., , ). The authors thus concluded that DLDH744 existed as a homodimer.…”

“…The activities of purified LDHs were assayed by monitoring the decrease in absorption at 340 nm (from NADH) with pyruvate as substrate, as described elsewhere (Zhu et al., , ). The reaction was initiated by enzyme addition.…”

“…In this pathway, lactate dehydrogenases (LDHs) play key roles by catalyzing not only the transformation of pyruvate to lactate, but also the oxidation of nicotinamide adenine dinucleotide (NADH), which constitutes an important step in the metabolism and energy conversion of living cells (Andreevskaya et al., ; Wang, Ingram, & Shanmugam, ). Two optically pure isomers of lactate were produced from pyruvate as separate entities through reactions catalyzed by either the chiral‐specific d ‐lactate dehydrogenase (D‐LDH, EC 1.1.1.28) or l ‐lactate dehydrogenase (L‐LDH, EC 1.1.1.27) (Singhvi, Jadhav, & Gokhale, ; Sun, Zhang, Lyu, Wang, & Yu, ; Wang, Cai, Zhu, Guo, & Yu, ; Zheng et al., ; Zhu et al., ). A sequence comparison has shown that D‐LDH and L‐LDH belong to two distinct families, the NAD‐dependent L‐ and D‐2‐hydroxyacid dehydrogenases, respectively (Cristescu & Egbosima, ; Ma et al., ).…”

“…On the other hand, the complete genome sequence of S. inulinus CASD was reported in 2010 (Yu et al., ). Meanwhile, a D‐LDH from this strain that could preferentially use both NADH and NADPH (nicotinamide adenine dinucleotide phosphate) as coenzymes was cloned and named DLDH744 (Zhu et al., ). More recent studies by Zhu, Xu, Wang, Dong, and Yu () confirmed that the above enzyme had both D‐LDH and glutamate dehydrogenase activities.…”

Contributory roles of two l-lactate dehydrogenases for l-lactic acid production in thermotolerant Bacillus coagulans

“…L-Lactate dehydrogenase (L-LDH; EC 1.1.1.27) and D-lactate dehydrogenase (D-LDH; EC 1.1.1.28) are responsible for the conversion of pyruvic acid (produced by glycolysis) to L- and D-lactic acid, respectively121314. Studies have shown that the relative catalytic efficiencies of ldhL - and ldhD -encoded products are crucial for the optical purity of lactic acid produced by Lactobacillus strains3.…”