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

8STD の概要

• エントリーDOI: 10.2210/pdb8std/pdb
• 分子名称: Pyridinium-3,5-biscarboxylic acid mononucleotide synthase, MAGNESIUM ION, NICOTINIC ACID ADENINE DINUCLEOTIDE (3 entities in total)
• 機能のキーワード: carboxylase, hydrolase, lyase (carbon-carbon), lyase
• 由来する生物種: Lactiplantibacillus plantarum WCFS1
• タンパク質・核酸の鎖数: 6
• 化学式量合計: 163176.61

構造登録者

Chatterjee, S.,Rankin, J.A.,Hu, J.,Hausinger, R.P. (登録日: 2023-05-10, 公開日: 2023-12-27)

主引用文献

Chatterjee, S.,Nevarez, J.L.,Rankin, J.A.,Hu, J.,Hausinger, R.P.
Structure of the LarB-Substrate Complex and Identification of a Reaction Intermediate during Nickel-Pincer Nucleotide Cofactor Biosynthesis.
Biochemistry, 62:3096-3104, 2023

PubMed Abstract: LarB 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 for substrate carboxylation and reported the structure of a 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 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 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..

PubMed: 37831946
DOI: 10.1021/acs.biochem.3c00242

実験手法

X-RAY DIFFRACTION (2.65 Å)