A distinct holoenzyme organization for two-subunit pyruvate carboxylase.

Philip H. Choi,Min-Han Lin,Liang Tong,Lars E. P. Dietrich,Jeanyoung Jo,Chi-Yuan Chou,Yu-Cheng Lin

doi:10.1038/ncomms12713

Abstract

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components. It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. In contrast, PC is a two-subunit enzyme in a collection of Gram-negative bacteria, with the α subunit containing the BC and the β subunit the CT and BCCP domains, and it is believed that the holoenzyme has α4β4 stoichiometry. We report here the crystal structures of a two-subunit PC from Methylobacillus flagellatus. Surprisingly, our structures reveal an α2β4 stoichiometry, and the overall architecture of the holoenzyme is strikingly different from that of the homo-tetrameric PCs. Biochemical and mutagenesis studies confirm the stoichiometry and other structural observations. Our functional studies in Pseudomonas aeruginosa show that its two-subunit PC is important for colony morphogenesis.

Highlights

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria
In a diverse array of Gram-negative bacteria among the phyla Proteobacteria and Aquificae (Supplementary Fig. 1), the PC enzyme is encoded by two separate genes, with the biotin carboxylase (BC) domain residing in a 52 kD a subunit and the CT and biotin carboxyl carrier protein (BCCP) domains in a 67 kD b subunit (Fig. 1a)[11]
In contrast, DPC and K572A colonies begin to wrinkle on day 2 and exhibit a flatter morphology with several exaggerated wrinkles in a ‘spoke’ formation, which is reminiscent of morphologies that arise during electron acceptor-limited conditions[32]

Summary

Introduction

Pyruvate carboxylase (PC) has important roles in metabolism and is crucial for virulence for some pathogenic bacteria. PC contains biotin carboxylase (BC), carboxyltransferase (CT) and biotin carboxyl carrier protein (BCCP) components It is a single-chain enzyme in eukaryotes and most bacteria, and functions as a 500 kD homo-tetramer. The structures reveal a new domain, named the PC tetramerization (PT) domain[9] or allosteric domain[8] that is important for tetramer formation by Staphylococcus aureus and human PCs (SaPC and HsPC) and for interaction with acetyl-CoA8–10. This domain is formed by two discontinuous segments of the protein—the linker between BC and CT and that between CT and BCCP (Fig. 1a). The two-subunit PC from Azotobacter vinelandii is necessary for growth on minimal media with glucose or sucrose as the sole carbon source, and a deletion mutant accumulates the compound poly-bhydroxybutyrate due to a slowdown of the TCA cycle[17,18]

Methods

Results

Conclusion