8std
S127A variant of LarB, a carboxylase/hydrolase involved in synthesis of the cofactor for lactate racemase, in complex with authentic substrate NaAD and soaked with CS2
Structural highlights
FunctionLARB_LACPL Involved in the biosynthesis of a nickel-pincer cofactor ((SCS)Ni(II) pincer complex). Carboxylates the pyridinium ring of nicotinic acid adenine dinucleotide (NaAD) and cleaves the phosphoanhydride bond to release AMP and generate pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN) (PubMed:27114550). LarB can hydrolyze NaAD directly or it first forms an adduct with NaAD that releases AMP and reacts with bicarbonate/CO2 to generate P2CMN (PubMed:27114550). Is required for the activation of the lactate racemase LarA (PubMed:24710389). May also be involved in the activation of other nickel-pincer cofactor-dependent enzymes (PubMed:27114550).[1] [2] Publication Abstract from PubMedLarB catalyzes the first step of biosynthesis for the nickel-pincer nucleotide cofactor by converting nicotinic acid adenine dinucleotide (NaAD) to AMP and pyridinium-3,5-biscarboxylic acid mononucleotide (P2CMN). Prior studies had shown that LarB uses CO(2) for substrate carboxylation and reported the structure of a Lactiplantibacillus plantarum LarB.NAD(+) complex, revealing a covalent linkage between Cys221 and C4 of the pyridine ring. This interaction was proposed to promote C5 carboxylation, with C5-carboxylated-NaAD suggested to activate magnesium-bound water, leading to phosphoanhydride hydrolysis. Here, we extended the analysis of wild-type LarB by using ultraviolet-visible spectroscopy to obtain additional evidence for cysteinyl side chain attachment to the ring of NAD(+), thus demonstrating that this linkage is not a crystallization artifact. Using the S127A variant of L. plantarum LarB, a form of the enzyme with a reduced rate of NaAD hydrolysis, we examined its interaction with the authentic substrate. The intermediate arising from C5 carboxylation of NaAD, dinicotinic acid adenine dinucleotide (DaAD), was identified by using mass spectrometry. S127A LarB exhibited spectroscopic evidence of a Cys221-NAD(+) adduct, but a covalent enzyme-NaAD linkage was not detectable. We determined the S127A LarB.NaAD structure, providing new insights into the enzyme mechanism, and tentatively identified the position and mode of CO(2) binding. The crystal structure revealed the location of the side chain for Glu180, which was previously disordered, but showed that it is not well positioned to abstract the C5 proton in the adduct species to restore aromaticity as Cys221 is expelled. Based on these combined results, we propose a revised catalytic mechanism of LarB.. Structure of the LarB-Substrate Complex and Identification of a Reaction Intermediate during Nickel-Pincer Nucleotide Cofactor Biosynthesis.,Chatterjee S, Nevarez JL, Rankin JA, Hu J, Hausinger RP Biochemistry. 2023 Nov 7;62(21):3096-3104. doi: 10.1021/acs.biochem.3c00242. Epub , 2023 Oct 13. PMID:37831946[3] From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine. References
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