Protein lysine de-2-hydroxyisobutyrylation by CobB in prokaryotes

Abstract

Lysine 2-hydroxyisobutyrylation (Khib) has recently been shown to be an evolutionarily conserved histone mark. Here, we report that CobB serves as a lysine de-2-hydroxyisobutyrylation enzyme that regulates glycolysis and cell growth in prokaryotes. We identified the specific binding of CobB to Khib using a novel self-assembled multivalent photocrosslinking peptide probe and demonstrated that CobB can catalyze lysine de-2-hydroxyisobutyrylation both in vivo and in vitro. R58 of CobB is a critical site for its de-2-hydroxyisobutyrylase activity. Using a quantitative proteomics approach, we identified 99 endogenous substrates that are targeted by CobB for de-2-hydroxyisobutyrylation. We further demonstrated that CobB can regulate the catalytic activities of enolase (ENO) by removing K343hib and K326ac of ENO simultaneously, which account for changes of bacterial growth. In brief, our study dissects a Khib-mediated molecular mechanism that is catalyzed by CobB for the regulation of the activity of metabolic enzymes as well as the cell growth of bacteria.

Fig. 1. Capture of the binding proteins of Khib.

(A) Structures of acetylation, succinylation, and 2-hydroxyisobutyrylation. (B) Workflow of the capture and enrichment of binding proteins of Khib by self-assembled multivalent photoaffinity peptide probes. UV, ultraviolet; AuNP, gold nanoparticles. (C) Volcano plot of enrichment result. The identified proteins with an abundance increase of more than twofold (log₂[S/C] > 1) and P value less than 0.05 were marked red.

Fig. 2. CobB catalyzes lysine de-2-hydroxyisobutyrylation in vivo.

(A) Western blotting (IB) analysis of lysine acylation in protein lysates from CobB-overexpressing P. mirabilis cells. (B) Western blotting analysis of lysine acylation in protein lysates from CobB-overexpressing E. coli BL21 (DE3) cells. (C) Western blotting analysis of lysine acylation in protein lysates from npdA KO E. coli MG1655 cells cultured in LB medium. (D) Western blotting analysis of lysine acylation in protein lysates from npdA KO E. coli MG1655 cells cultured in M9 medium.

Fig. 3. Characterization of de-2-hydroxyisobutyrylation of CobB.

(A) Multiple sequence alignment of CobB from different species by Clustal×2. (B) ITC analysis of the affinity of recombinant CobB (WT) and mutated CobB (Y55F and R58M) with the 2-hydroxyisobutyrylated peptide [YGDEQVK(hib)QWR]. (C) De-2-hydroxyisobutyrylation kinetic parameters (k_cat, K_m, and k_cat/K_m) of WT and mutated CobB (Y55F and R58M). ND means “not determined” because of a lack of enzyme saturation with the highest substrate concentration used. (D) Western blotting analysis of Khib in protein lysates from npdA KO E. coli MG1655 cells overexpressed with pTrc99a, CobB, and CobB-mutated R95M and cultured in M9 medium.

Fig. 4. CobB catalyzes lysine de-2-hydroxyisobutyrylation in vitro.

(A) Western blotting showed that CobB can catalyze in vitro de-2-hydroxyisobutyrylation, deacetylation, and desuccinylation reactions in whole P. mirabilis cell proteins. (B to D) LC-MS detection of lysine de-2-hydroxyisobutyrylation, deacetylation, and desuccinylation activities of CobB in peptide substrates. Synthetic peptides [YGDEQVK(hib)QWR, YGDEQVK(ac)QWR, and YGDEQVK(su)QWR, from gpmA of P. mirabilis] were used as the substrates. The assays were carried out without CobB, with CobB, without NAD⁺, and with CobB in the presence of NAM (10 mM). A triangle and a circle indicate modified and unmodified peptides, respectively.

Fig. 5. Profiling of Kac, Ksucc, and Khib in P. mirabilis.

(A) Statistical analysis of the Kac, Ksucc, and Khib proteins, peptides, and sites. (B) Statistical analysis of the common acylated proteins. (C) Statistical analysis of the common acylated sites. (D) The histograms show experimentally determined relative protein abundance distributions for the samples used to analyze 2-hydroxyisobutyrylation. (E) The scatterplots show the ratio of Khib peptides in CobB KO versus WT E. coli cells in relation to average peptide intensities. (F) The bar graph shows pathways enriched in CobB-regulated Khib proteomes. (G) The list shows the carbon metabolic enzymes with a significant up-regulation of the Khib level under CobB KO conditions.

Fig. 6. CobB regulates ENO activity and influences cell growth by erasing Khib and Kac.

(A) Multiple sequence alignment of ENO from different species by Clustal×2. (B) Identification of K326ac and K343hib in ENO. (C) Detection of the activity of ENO WT and its mutants (K343T, K326Q, and K326Q/K343T) (data are means ± SEM from three independent assays; *P < 0.05, **P < 0.001). (D) Western blotting determined the Khib, Kac, and Ksucc levels of purified recombinant ENO. (E) Western blotting revealed that NAD-dependent CobB can catalyze de-2-hydroxyisobutyrylation/deacetylation/desuccinylation with ENO. ENO was incubated with CobB in the presence of NAD⁺ (0.5 mM) for 10 hours at 25°C. NAM (10 mM) was added to the reaction as a CobB inhibitor. (F) LC-MS detection of lysine de-2-hydroxyisobutyrylation and deacetylation activities of CobB with ENO peptides [GIANSILI-K(hib)FNQIGSLTETLAAIK, IQLVGDDLFVTNTK(Ac)ILK]. (G) Comparison of the quantities of K326ac and K343hib in T vector and CobB-overexpressing P. mirabilis (data are presented as means ± SEM, and P values were determined by two-tailed Student’s t test; NS, not significant; P > 0.05, *P < 0.05, **P < 0.01, ***P < 0.001). (H) Measurement of the PEP amounts of T vector and CobB-overexpressing P. mirabilis (data are presented as means ± SEM; P values were determined by two-tailed Student’s t test; **P < 0.01). (I) Measurement of the PEP amounts of ENO-overexpressing P. mirabilis that transformed ENO WT and mutation (K326Q, K343T, and K326Q/K343T) vectors (data are means ± SEM from three independent assays; *P < 0.05, **P < 0.001). (J) Measurement growth curve of ENO-overexpressing P. mirabilis that transformed ENO WT and mutation (K326Q, K343T, and K326Q/K343T) vectors (data are means ± SEM from three independent assays, *P < 0.05). OD₆₀₀, optical density at 600 nm. (K) Graphic model as discussed in the text. CobB can promote bacterial growth by effective de-2-hydroxyisobutyrylation of K343 and deacetylation of K326 of ENO. 2PG, 2-phosphoglycerate.