Heidi A. Crosby

System-wide Studies of N-Lysine Acetylation in Rhodopseudomonas palustris Reveal Substrate Specificity of Protein Acetyltransferases

Background: Protein acetylation is widespread in prokaryotes. Results: Six new enzymes whose activities are controlled by acetylation were identified, and their substrate preferences were established. A new protein acetyltransferase was also identified, and its substrate specificity was determined. Conclusion: Protein acetyltransferases acetylate a conserved lysine residue in protein substrates. Significance: Protein acetyltransferases acetylate AMP-forming acyl-CoA synthetases and regulate fatty acid metabolism. N-Lysine acetylation is a posttranslational modification that has been well studied in eukaryotes and is likely widespread in prokaryotes as well. The central metabolic enzyme acetyl-CoA synthetase is regulated in both bacteria and eukaryotes by acetylation of a conserved lysine residue in the active site. In the purple photosynthetic α-proteobacterium Rhodopseudomonas palustris, two protein acetyltransferases (RpPat and the newly identified RpKatA) and two deacetylases (RpLdaA and RpSrtN) regulate the activities of AMP-forming acyl-CoA synthetases. In this work, we used LC/MS/MS to identify other proteins regulated by the N-lysine acetylation/deacetylation system of this bacterium. Of the 24 putative acetylated proteins identified, 14 were identified more often in a strain lacking both deacetylases. Nine of these proteins were members of the AMP-forming acyl-CoA synthetase family. RpPat acetylated all nine of the acyl-CoA synthetases identified by this work, and RpLdaA deacetylated eight of them. In all cases, acetylation occurred at the conserved lysine residue in the active site, and acetylation decreased activity of the enzymes by >70%. Our results show that many different AMP-forming acyl-CoA synthetases are regulated by N-lysine acetylation. Five non-acyl-CoA synthetases were identified as possibly acetylated, including glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and Rpa1177, a putative 4-oxalocrotonate tautomerase. Neither RpPat nor RpKatA acetylated either of these proteins in vitro. It has been reported that Salmonella enterica Pat (SePat) can acetylate a number of metabolic enzymes, including GAPDH, but we were unable to confirm this claim, suggesting that the substrate range of SePat is not as broad as suggested previously.

Reversible Nε‐lysine acetylation regulates the activity of acyl‐CoA synthetases involved in anaerobic benzoate catabolism in Rhodopseudomonas palustris

Rhodopseudomonas palustris grows photoheterotrophically on aromatic compounds available in aquatic environments rich in plant‐derived lignin. Benzoate degradation is regulated at the transcriptional level in R. palustris in response to anoxia and the presence of benzoate and/or benzoyl‐CoA (Bz‐CoA). Here, we report evidence that anaerobic benzoate catabolism in this bacterium is also regulated at the post‐translational level. In this pathway, benzoate is activated to Bz‐CoA by the AMP‐forming Bz‐CoA synthetase (BadA) enzyme. Mass spectrometry and mutational analysis data indicate that residue Lys512 is critical to BadA activity. Acetylation of Lys512 inactivated BadA; deacetylation reactivated BadA. Likewise, 4‐hydroxybenzoyl‐CoA (HbaA) and cyclohexanecarboxyl‐CoA (AliA) synthetases were also reversibly acetylated. We identified one acetyltransferase that modified BadA, Hba and AliA in vitro. The acetyltransferase enzyme is homologous to the protein acetyltransferase (Pat) enzyme of Salmonella enterica sv Typhimurium LT2, thus we refer to it as RpPat. RpPat also modified acetyl‐CoA (Ac‐CoA) synthetase (Acs) from R. palustris. In vivo data indicate that at least two deacetylases reactivate BadAAc. One is SrtN (encoded by srtN, formerly rpa2524), a sirtuin‐type NAD+‐dependent deacetylase (O‐acetyl‐ADPribose‐forming); the other deacetylase is LdaA (encoded by ldaA, for lysine deacetylase A; formerly rpa0954), an acetate‐forming protein deacetylase. LdaA reactivated HbaAc and AliAAcin vitro.

Identification of the Biosynthetic Gene Cluster and an Additional Gene for Resistance to the Antituberculosis Drug Capreomycin

ABSTRACT Capreomycin (CMN) belongs to the tuberactinomycin family of nonribosomal peptide antibiotics that are essential components of the drug arsenal for the treatment of multidrug-resistant tuberculosis. Members of this antibiotic family target the ribosomes of sensitive bacteria and disrupt the function of both subunits of the ribosome. Resistance to these antibiotics in Mycobacterium species arises due to mutations in the genes coding for the 16S or 23S rRNA but can also arise due to mutations in a gene coding for an rRNA-modifying enzyme, TlyA. While Mycobacterium species develop resistance due to alterations in the drug target, it has been proposed that the CMN-producing bacterium, Saccharothrix mutabilis subsp. capreolus, uses CMN modification as a mechanism for resistance rather than ribosome modification. To better understand CMN biosynthesis and resistance in S. mutabilis subsp. capreolus, we focused on the identification of the CMN biosynthetic gene cluster in this bacterium. Here, we describe the cloning and sequence analysis of the CMN biosynthetic gene cluster from S. mutabilis subsp. capreolus ATCC 23892. We provide evidence for the heterologous production of CMN in the genetically tractable bacterium Streptomyces lividans 1326. Finally, we present data supporting the existence of an additional CMN resistance gene. Initial work suggests that this resistance gene codes for an rRNA-modifying enzyme that results in the formation of CMN-resistant ribosomes that are also resistant to the aminoglycoside antibiotic kanamycin. Thus, S. mutabilis subsp. capreolus may also use ribosome modification as a mechanism for CMN resistance.

In Salmonella enterica, the sirtuin‐dependent protein acylation/deacylation system (SDPADS) maintains energy homeostasis during growth on low concentrations of acetate

Acetyl‐coenzyme A synthetase (Acs) activates acetate into acetyl‐coenzyme A (Ac‐CoA) in most cells. In Salmonella enterica, acs expression and Acs activity are controlled. It is unclear why the sirtuin‐dependent protein acylation/deacylation system (SDPADS) controls the activity of Acs. Here we show that, during growth on 10 mM acetate, acs+ induction in a S. enterica strain that cannot acetylate (i.e. inactivate) Acs leads to growth arrest, a condition that correlates with a drop in energy charge (0.17) in the acetylation‐deficient strain, relative to the energy charge in the acetylation‐proficient strain (0.71). Growth arrest was caused by elevated Acs activity, a conclusion supported by the isolation of a single‐amino‐acid variant (AcsG266S), whose overproduction did not arrest growth. Acs‐dependent depletion of ATP, coupled with the rise in AMP levels, prevented the synthesis of ADP needed to replenish the pool of ATP. Consistent with this idea, overproduction of ADP‐forming Ac‐CoA‐synthesizing systems did not affect the growth behaviour of acetylation‐deficient or acetylation‐proficient strains. The AcsG266S variant was > 2 orders of magnitude less efficient than the AcsWT enzyme, but still supported growth on 10 mM acetate. This work provides the first evidence that SDPADS function helps cells maintain energy homeostasis during growth on acetate.

Structure-Guided Expansion of the Substrate Range of Methylmalonyl Coenzyme A Synthetase (MatB) of Rhodopseudomonas palustris

ABSTRACT Malonyl coenzyme A (malonyl-CoA) and methylmalonyl-CoA are two of the most commonly used extender units for polyketide biosynthesis and are utilized to synthesize a vast array of pharmaceutically relevant products with antibacterial, antiparasitic, anticholesterol, anticancer, antifungal, and immunosuppressive properties. Heterologous hosts used for polyketide production such as Escherichia coli often do not produce significant amounts of methylmalonyl-CoA, however, requiring the introduction of other pathways for the generation of this important building block. Recently, the bacterial malonyl-CoA synthetase class of enzymes has been utilized to generate malonyl-CoA and methylmalonyl-CoA directly from malonate and methylmalonate. We demonstrate that in the purple photosynthetic bacterium Rhodopseudomonas palustris, MatB (RpMatB) acts as a methylmalonyl-CoA synthetase and is required for growth on methylmalonate. We report the apo (1.7-Å resolution) and ATP-bound (2.0-Å resolution) structure and kinetic analysis of RpMatB, which shows similar activities for both malonate and methylmalonate, making it an ideal enzyme for heterologous polyketide biosynthesis. Additionally, rational, structure-based mutagenesis of the active site of RpMatB led to substantially higher activity with ethylmalonate and butylmalonate, demonstrating that this enzyme is a prime target for expanded substrate specificity.

Structural Insights into the Substrate Specificity of the Rhodopseudomonas palustris Protein Acetyltransferase RpPat: IDENTIFICATION OF A LOOP CRITICAL FOR RECOGNITION BY RpPat*♦

Background: RpPat acetylates many acyl-CoA synthetase enzymes. Results: RpPat does not acetylate RpMatB but can acetylate chimeric versions of it that differ from the wild-type enzyme in a loop region >20 Å away from the acetylated lysine. Conclusion: RpPat likely interacts with a large surface area of its substrates. Significance: RpPat substrates cannot be predicted by a short acetylation motif alone. Lysine acetylation is a post-translational modification that is important for the regulation of metabolism in both prokaryotes and eukaryotes. In bacteria, the best studied protein acetyltransferase is Pat. In the purple photosynthetic bacterium Rhodopseudomonas palustris, at least 10 AMP-forming acyl-CoA synthetase enzymes are acetylated by the Pat homologue RpPat. All bona fide RpPat substrates contain the conserved motif PX4GK. Here, we show that the presence of such a motif is necessary but not sufficient for recognition by RpPat. RpPat failed to acetylate the methylmalonyl-CoA synthetase of this bacterium (hereafter RpMatB) in vivo and in vitro, despite the homology of RpMatB to known RpPat substrates. We used RpMatB to identify structural determinants that are recognized by RpPat. To do this, we constructed a series of RpMatB chimeras that became substrates of RpPat. In such chimeras, a short region (11–25 residues) of RpMatB located >20 residues N-terminal to the acetylation site was replaced with the corresponding sequences from other AMP-forming acyl-CoA synthetases that were known RpPat substrates. Strikingly, the enzymatic activity of RpMatB chimeras was regulated by acetylation both in vitro and in vivo. Crystal structures of two of these chimeras showed that the major difference between them and wild-type RpMatB was within a loop region ∼23 Å from the acetylation site. On the basis of these results, we suggest that RpPat likely interacts with a relatively large surface of its substrates, in addition to the PX4GK motif, and that RpPat probably has relatively narrow substrate specificity.