The development of biotechnological lactic acid production has attracted attention to the potential production of an optically pure isomer of lactic acid, although the relationship between fermentation and the biosynthesis of highly optically pure D-lactic acid remains poorly understood. Sporolactobacillus terrae SBT-1 is an excellent D-lactic acid producer that depends on cultivation conditions. Herein, three enzymes responsible for synthesizing optically pure D-lactic acid, including D-lactate dehydrogenase (D-LDH; encoded by ldhDs), L-lactate dehydrogenase (L-LDH; encoded by ldhLs), and lactate racemase (Lar; encoded by larA), were quantified under different organic nitrogen sources and concentration to study the relationship between fermentation conditions and synthesis pathway of optically pure lactic acid. Different organic nitrogen sources and concentrations significantly affected the quantity and quality of D-lactic acid produced by strain SBT-1 as well as the synthetic optically pure lactic acid pathway. Yeast extract is a preferred organic nitrogen source for achieving high catalytic efficiency of D-lactate dehydrogenase and increasing the transcription level of ldhA2, indicating that this enzyme plays a major role in D-lactic acid formation in S. terrae SBT-1. Furthermore, lactate racemization activity could be regulated by the presence of D-lactic acid. The results of this study suggest that specific nutrient requirements are necessary to achieve a stable and highly productive fermentation process for the D-lactic acid of an individual strain.

Lactic acid bacteria (LAB) present various benefits to humans; they play key roles in the fermentation of food and as probiotics. Acidic conditions are common to both LAB in the intestinal tract as well as fermented foods. Lactiplantibacillus plantarum is a facultative homofermentative bacterium, and lactic acid is the end metabolite of glycolysis. To characterize how L. plantarum responds to lactic acid, we investigated its transcriptome following treatment with hydrochloride (HCl) or dl-lactic acid at an early stage of growth. Bacterial growth was more attenuated in the presence of lactic acid than in the presence of HCl at the same pH range. Bacterial transcriptome analysis showed that the expression of 67 genes was significantly altered (log2FC > 2 or < 2). A total of 31 genes were up- or downregulated under both conditions: 19 genes in the presence of HCl and 17 genes in the presence of dl-lactic acid. The fatty acid synthesis-related genes were upregulated in both acidic conditions, whereas the lactate racemization-related gene (lar) was only upregulated following treatment with dl-lactic acid. In particular, lar expression increased following l-lactic acid treatment but did not increase following HCl or d-lactic acid treatment. Expression of lar and production of d-lactic acid were investigated with malic and acetic acid; the results revealed a higher expression of lar and production of d-lactic acid in the presence of malic acid than that in the presence of acetic acid.

Cofactors are essential components of numerous enzymes, therefore their characterization by structural, biophysical, and biochemical approaches is crucial for understanding the resulting catalytic and regulatory mechanisms. In this chapter, we present a case study of a recently discovered cofactor, the nickel-pincer nucleotide (NPN), by demonstrating how we identified and thoroughly characterized this unprecedented nickel-containing coenzyme that is tethered to lactase racemase from Lactiplantibacillus plantarum. In addition, we describe how the NPN cofactor is biosynthesized by a panel of proteins encoded in the lar operon and describe the properties of these novel enzymes. Comprehensive protocols for conducting functional and mechanistic studies of NPN-containing lactate racemase (LarA) and the carboxylase/hydrolase (LarB), sulfur transferase (LarE), and metal insertase (LarC) used for NPN biosynthesis are provided for potential applications towards characterizing enzymes in the same or homologous families.

Inspired by the catalytic mechanism and active site structure of lactate racemase, three scorpion-like SCS nickel pincer complexes were proposed as potential catalysts for transfer hydrogenation of ketones and imines with ammonia-borane (AB) as the hydrogen source. Density functional theory calculations reveal a stepwise hydride and proton transfer mechanism for the dehydrocoupling of AB and hydrogenation of N-methylacetonimine, and a concerted proton-coupled hydride transfer process for hydrogenation of acetone, acetophenone, and 3-methyl-2-butanone. Among all proposed Ni complexes, the one with symmetric NH2 group on both arms of the SCS pincer ligand has the lowest free energy barrier of 15.0 kcal/mol for dehydrogenation of AB, as well as total free energy barriers of 17.8, 18.2, 18.0, and 18.6 kcal/mol for hydrogenation of acetone, N-methylacetonimine, acetophenone, and 3-methyl-2-butanone, respectively.

Bacterial lactate racemase is a nickel-dependent enzyme that contains a cofactor, nickel pyridinium-3,5-bisthiocarboxylic acid mononucleotide, hereafter named nickel-pincer nucleotide (NPN). The LarC enzyme from the bacterium Lactobacillus plantarum participates in NPN biosynthesis by inserting nickel ion into pyridinium-3,5-bisthiocarboxylic acid mononucleotide. This reaction, known in organometallic chemistry as a cyclometalation, is characterized by the formation of new metal-carbon and metal-sulfur σ bonds. LarC is therefore the first cyclometallase identified in nature, but the molecular mechanism of LarC-catalyzed cyclometalation is unknown. Here, we show that LarC activity requires Mn2+-dependent CTP hydrolysis. The crystal structure of the C-terminal domain of LarC at 1.85 Å resolution revealed a hexameric ferredoxin-like fold and an unprecedented CTP-binding pocket. The loss-of-function of LarC variants with alanine variants of acidic residues leads us to propose a carboxylate-assisted mechanism for nickel insertion. This work also demonstrates the in vitro synthesis and purification of the NPN cofactor, opening new opportunities for the study of this intriguing cofactor and of NPN-utilizing enzymes.

Fermentative production of optically pure lactic acid (LA) has attracted great interest because of the increased demand for plant-based plastics. For cost-effective LA production, an engineered Lactobacillus plantarum NCIMB 8826 strain, which enables the production of optically pure l-LA from raw starch, is constructed. The wild-type strain produces a racemic mixture of d- and l-LA from pyruvate by the action of the respective lactate dehydrogenases (LDHs). Therefore, the gene encoding D-LDH (ldhD) is deleted. Although no decrease in d-LA formation is observed in the ΔldhD mutant, additional disruption of the operon encoding lactate racemase (larA-E), which catalyzes the interconversion between d- and l-LA, completely abolished d-LA production. From 100 g L-1 glucose, the ΔldhD ΔlarA-E mutant produces 87.0 g L-1 of l-LA with an optical purity of 99.4%. Subsequently, a plasmid is introduced into the ΔldhD ΔlarA-E mutant for the secretion of α-amylase from Streptococcus bovis 148. The resulting strain could produce 50.3 g L-1 of l-LA from raw corn starch with a yield of 0.91 (g per g of consumed sugar) and an optical purity of 98.6%. The engineered L. plantarum strain would be useful in the production of l-LA from starchy materials.

Analysis of lactate racemase (Lar) in lactic acid bacteria (LAB) has been a scientific challenge for many years, as indicated by the numerous contradictory reports on this activity. Recently, genetic and biochemical studies of the Lar system of Lactobacillus plantarum have unveiled the complexity of this particular enzymatic system. Lar activity is associated with LarA and its nickel-containing cofactor, synthesized from nicotinic acid adenine dinucleotide by the three biosynthetic enzymes: LarB, LarC, and LarE. In addition to these core Lar enzymes, a nickel transporter (Lar(MN)QO), a lactic acid channel (LarD) and a transcriptional regulator (LarR) which promotes expression of the lar genes in the presence of excess L-lactate are also part of the Lar system of Lb. plantarum and of many other LAB. These proteins promote racemization of external L-lactate, in addition to carrying out intracellular racemization. This additional outcome suggests that racemization of L-lactate is not only required for cell wall biosynthesis, as reported before, but may have additional roles in lactate production and utilization in LAB. Finally, bioinformatics analyses indicate that some Lar homologs probably catalyze reactions other than lactate racemization.

QM/MM calculations reveal that the nickel pincer complex in lactate racemase functions as a reversible \"single-center electrode\" that accepts and donates back an electron. In this way, it catalyzes the isomerization process d-lactate⇌l-lactate through successive proton-coupled electron-transfer steps.

Lactate racemase is the first enzyme known to possess a metal pincer active site. The enzyme interconverts d- and l-lactic acid, which is important for the assembly of cell walls in many microorganisms. Here, we report a synthetic model of the active site of lactate racemase, which features a pyridinium-based SCS pincer ligand framework bound to nickel. The model complex mediates the dehydrogenation of alcohols, a reaction relevant to lactate racemization. Experimental and computational data indicate ligand participation in the dehydrogenation reaction.

Recently, a lactate racemase was discovered as a new Ni-dependent enzyme with a unique tethered NAD-like cofactor. We report the first computational study aimed at deciphering the previously unclear role of the Ni-tethered cofactor in reactions of the lactate racemase. Our calculations revealed that the cofactor increases the dehydrogenation barriers. The formation of a metastable NADH-like pyruvate intermediate and two nearby histidine bases are proposed as the key factors in the racemization reaction. Such destabilization of intermediates by the cofactor is uncommon in enzymatic catalysis. This result provides new insight into the design of a reactive metal-tethered NADH-like complex for synthetic hydrogenations.