Nickel-pincer cofactor biosynthesis involves LarB-catalyzed pyridinium carboxylation and LarE-dependent sacrificial sulfur insertion.
The lactate racemase enzyme (LarA) of Lactobacillus plantarum harbors a (SCS)Ni(II) pincer complex derived from nicotinic acid. Synthesis of the enzyme-bound cofactor requires LarB, LarC, and LarE, which are widely distributed in microorganisms. The functions of the accessory proteins are unknown, but the LarB C terminus resembles aminoimidazole ribonucleotide carboxylase/mutase, LarC binds Ni and could act in Ni delivery or storage, and LarE is a putative ATP-using enzyme of the pyrophosphatase-loop superfamily. Here, we show that LarB carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP. The resulting biscarboxylic acid intermediate is transformed into a bisthiocarboxylic acid species by two single-turnover reactions in which sacrificial desulfurization of LarE converts its conserved Cys176 into dehydroalanine. Our results identify a previously unidentified metabolic pathway from NaAD using unprecedented carboxylase and sulfur transferase reactions to form the organic component of the (SCS)Ni(II) pincer cofactor of LarA. In species where larA is absent, this pathway could be used to generate a pincer complex in other enzymes.
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824; Institute of Life Sciences, Université catholique de Louvain, B-1348 Louvain-La-Neuve, Belgium.
Benoît
B
Desguin
Institute of Life Sciences, Université catholique de Louvain, B-1348 Louvain-La-Neuve, Belgium.
Patrice
P
Soumillion
Institute of Life Sciences, Université catholique de Louvain, B-1348 Louvain-La-Neuve, Belgium.
Pascal
P
Hols
Department of Microbiology and Molecular Genetics, Michigan State University, East Lansing, MI 48824; hausinge@msu.edu.
Robert P
0000-0002-3643-2054
RP
Hausinger
D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000502:physiology; D048191:Lactiplantibacillus plantarum / Q000201:enzymology*; D053858:Metabolic Networks and Pathways; D009243:NAD / Q000031:analogs & derivatives* / Q000378:metabolism; D009532:Nickel / Q000378:metabolism*; D019998:Racemases and Epimerases / Q000502:physiology*; D013455:Sulfur / Q000378:metabolism*
D009243:NAD; C018348:nicotinic acid adenine dinucleotide; D013455:Sulfur; D009532:Nickel; D002262:Carboxy-Lyases; C022378:phosphoribosylaminoimidazole carboxylase; D019998:Racemases and Epimerases; C021498:lactate racemase
lactate; nicotinic acid dinucleotide; racemase; sacrificial enzyme