The Structure of the PanD/PanZ Protein Complex Reveals Negative Feedback Regulation of Pantothenate Biosynthesis by Coenzyme A

Monteiro, Diana C. F.; Patel, Vijay; Bartlett, Christopher P.; Nozaki, Sukekatsu; Grant, Thomas D.; Gowdy, James; Thompson, Gary S.; Kalverda, Arnout P.; Snell, Edward H.; Niki, Hironori; Pearson, Arwen R.; Webb, Michael E.

doi:10.1016/j.chembiol.2015.03.017

Cited by 31 publications

(43 citation statements)

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“…5 ). Interestingly, the C-terminal portion of PanD identified by Monteiro et al [ 17 ] was one of the possible PanM binding regions identified in our study. Specifically, variations in the motif Ala118 to Ala126 identified by the bar in Fig.…”

Section: Resultssupporting

confidence: 55%

“…These residues form the predicted PanD binding site. The asterisk indicates identifies residue Asn45, which was found to be required for PanM binding to pro-PanD [ 17 ]. The bar atop the sequence on the lower panel spans residues Leu66-Gly76, which was also identified by structural studies to be required for PanZ/PanM:PanD interactions [ 17 ].…”

Section: Resultsmentioning

confidence: 99%

“…During the review of the work reported herein, Monteiro et al published the structure of E. coli PanD in complex with PanM (PanZ in E. coli ). This elegant and thorough set of studies revealed, among other things, the interaction domain between the PanD zymogen and PanM [ 17 ]. Their analysis established that PanM binds to the C-terminus of PanD.…”

Section: Resultsmentioning

confidence: 99%

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Phylogenetic and amino acid conservation analyses of bacterial l-aspartate-α-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain

et al. 2015

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BackgroundAll organisms must synthesize the enzymatic cofactor coenzyme A (CoA) from the precursor pantothenate. Most bacteria can synthesize pantothenate de novo by the condensation of pantoate and β-alanine. The synthesis of β-alanine is catalyzed by l-aspartate-α-decarboxylase (PanD), a pyruvoyl enzyme that is initially synthesized as a zymogen (pro-PanD). Active PanD is generated by self-cleavage of pro-PanD at Gly24-Ser25 creating the active-site pyruvoyl moiety. In Salmonella enterica, this cleavage requires PanM, an acetyl-CoA sensor related to the Gcn5-like N-acetyltransferases. PanM does not acetylate pro-PanD, but the recent publication of the three-dimensional crystal structure of the PanM homologue PanZ in complex with the PanD zymogen of Escherichia coli provides validation to our predictions and provides a framework in which to further examine the cleavage mechanism. In contrast, PanD from bacteria lacking PanM efficiently cleaved in the absence of PanM in vivo.ResultsUsing phylogenetic analyses combined with in vivo phenotypic investigations, we showed that two classes of bacterial l-aspartate-α-decarboxylases exist. This classification is based on their posttranslational activation by self-cleavage of its zymogen. Class I l-aspartate-α-decarboxylase zymogens require the acetyl-CoA sensor PanM to be cleaved into active PanD. This class is found exclusively in the Gammaproteobacteria. Class II l-aspartate-α-decarboxylase zymogens self cleave efficiently in the absence of PanM, and are found in a wide number of bacterial phyla. Several members of the Euryarchaeota and Crenarchaeota also contain Class II l-aspartate-α-decarboxylases. Phylogenetic and amino acid conservation analyses of PanM revealed a conserved region of PanM distinct from conserved regions found in related Gcn5-related acetyltransferase enzymes (Pfam00583). This conserved region represents a putative domain for interactions with l-aspartate-α-decarboxylase zymogens. This work may inform future biochemical and structural studies of pro-PanD-PanM interactions.ConclusionsExperimental results indicate that S. enterica and C. glutamicuml-aspartate-α-decarboxylases represent two different classes of homologues of these enzymes. Class I homologues require PanM for activation, while Class II self cleave in the absence of PanM. Computer modeling of conserved amino acids using structure coordinates of PanM and l-aspartate-α-decarboxylase available in the protein data bank (RCSB PDB) revealed a putative site of interactions, which may help generate models to help understand the molecular details of the self-cleavage mechanism of l-aspartate-α-decarboxylases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13104-015-1314-6) contains supplementary material, which is available to authorized users.

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Section: Resultssupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

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Phylogenetic and amino acid conservation analyses of bacterial l-aspartate-α-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain

et al. 2015

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“…PanD is produced as a zymogen (proPanD) that is activated by the PanZ·RCoA complex but is also inhibited by the same complex. 8 RCoA = AcCoA or CoA.…”

mentioning

confidence: 99%

“…We have recently reported the structure of the complex formed between the zymogen of E. coli aspartate α-decarboxylase (proPanD) and its regulatory protein, PanZ. 8 PanD is responsible for the production of β-alanine in the pantothenate biosynthesis pathway, 9 and its catalytic action is dependent upon formation of a covalently bound pyruvoyl group from a serine residue via rearrangement of its peptide backbone. 10 PanZ is required for the in vivo activation of E. coli proPanD, as the uncatalyzed rearrangement is too slow to support growth.…”

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confidence: 99%

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD–PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan)

et al. 2017

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The antimetabolite pentyl pantothenamide has broad spectrum antibiotic activity but exhibits enhanced activity against Escherichia coli. The PanDZ complex has been proposed to regulate the pantothenate biosynthetic pathway in E. coli by limiting the supply of β-alanine in response to coenzyme A concentration. We show that formation of such a complex between activated aspartate decarboxylase (PanD) and PanZ leads to sequestration of the pyruvoyl cofactor as a ketone hydrate and demonstrate that both PanZ overexpression-linked β-alanine auxotrophy and pentyl pantothenamide toxicity are due to formation of this complex. This both demonstrates that the PanDZ complex regulates pantothenate biosynthesis in a cellular context and validates the complex as a target for antibiotic development.

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Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

Arnott,

Morgan,

Hollingsworth

et al. 2024

Angewandte Chemie

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Quantitative and selective labelling of proteins is widely used in both academic and industrial laboratories, and catalytic labelling of proteins using transpeptidases such as sortases has proved to be a popular strategy for selective modification. A major challenge for this class of enzymes is that the majority of procedures require an excess of the labelling reagent or, alternatively, activated substrates rather than simple commercially‐sourced peptides. We report the use of a coupled enzyme strategy which enables quantitative N‐ and C‐terminal labelling of proteins using un‐activated labelling peptides. The use of an aminopeptidase in conjunction with a transpeptidase allows sequence‐specific degradation of the peptide by‐product, shifting the equilibriums to favor product formation which greatly enhances the reaction efficiency. Subsequent optimization of the reaction allows N‑terminal labelling of proteins using essentially equimolar ratios of peptide label to protein and C‐terminal labelling with only a small excess. Minimizing the amount of substrate required for quantitative labelling has the potential to improve industrial processes and facilitate the use of transpeptidation as a method for protein labelling.

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The Structure of the PanD/PanZ Protein Complex Reveals Negative Feedback Regulation of Pantothenate Biosynthesis by Coenzyme A

Cited by 31 publications

References 56 publications

Phylogenetic and amino acid conservation analyses of bacterial l-aspartate-α-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain

Phylogenetic and amino acid conservation analyses of bacterial l-aspartate-α-decarboxylase and of its zymogen-maturation protein reveal a putative interaction domain

The Mechanism of Regulation of Pantothenate Biosynthesis by the PanD–PanZ·AcCoA Complex Reveals an Additional Mode of Action for the Antimetabolite N-Pentyl Pantothenamide (N5-Pan)

Quantitative N‐ or C‐Terminal Labelling of Proteins with Unactivated Peptides by Use of Sortases and a d‐Aminopeptidase

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