Formyl-coenzyme A (CoA):oxalate CoA-transferase from the acidophile Acetobacter aceti has a distinctive electrostatic surface and inherent acid stability

Abstract

Bacterial formyl-CoA:oxalate CoA-transferase (FCOCT) and oxalyl-CoA decarboxylase work in tandem to perform a proton-consuming decarboxylation that has been suggested to have a role in generalized acid resistance. FCOCT is the product of uctB in the acidophilic acetic acid bacterium Acetobacter aceti. As expected for an acid-resistance factor, UctB remains folded at the low pH values encountered in the A. aceti cytoplasm. A comparison of crystal structures of FCOCTs and related proteins revealed few features in UctB that would distinguish it from nonacidophilic proteins and thereby account for its acid stability properties, other than a strikingly featureless electrostatic surface. The apparently neutral surface is a result of a "speckled" charge decoration, in which charged surface residues are surrounded by compensating charges but do not form salt bridges. A quantitative comparison among orthologs identified a pattern of residue substitution in UctB that may be a consequence of selection for protein stability by constant exposure to acetic acid. We suggest that this surface charge pattern, which is a distinctive feature of A. aceti proteins, creates a stabilizing electrostatic network without stiffening the protein or compromising protein-solvent interactions.

Figure 1

Western blot to detect protein expression in A. aceti strain 1023 cell lysates, using a primary antibody raised against a peptide with the UctB N-terminal sequence. The bands visualized correspond to UctB (two closely spaced bands at 47 and 48 kDa) and an unidentified 59 kDa protein. Lanes marked with an L are from the first logarithmic growth phase, associated with the conversion of ethanol to acetic acid. Lanes marked with an S were harvested during the first stationary phase. The initial ethanol level of the YPDE cultures is indicated above each pair of lanes. A lane containing pure H6UctB (2 kDa larger than native UctB) was used as a standard. Each lane was loaded with 5 μg total protein. M indicates the position of size standards (in kDa).

Figure 2

Thermal denaturation of H6UctB (filled circles) and H6YfdW (open circles) monitored by CD as a function of pH. T_m values were determined using changes in molar ellipticity at 222 nm, corresponding to loss of helical structure. The lines connect average T_m values at each pH for H6UctB (solid line) and H6YfdW (dotted lines); H6UctB duplicates at pH 4.5 and 7.0 have completely overlapping symbols. The arrow at pH 4.0 for H6YfdW indicates a maximum for a partially observed melting transition. At pH 3.5, H6YfdW melted completely during equilibration at 20°C. Full CD spectra (recorded at 20°C) and thermal unfolding profiles are given as Figures S4–7 of the Supporting Information.

Figure 3

Structure of H6UctB bound to CoA (PDB id 3ubm). A: Elements from both subunits make up the H6UctB active sites, each occupied by CoA (ball-and-stick rendering). Secondary structure elements are labelled; a topology diagram is provided as Figure S8 in the Supporting Information. B: Electrostatic surface potential of H6UctB (subunits A/B) viewed in the same orientation as panel A. Positive and negative regions were scaled as in panel C. Additional orientations are shown in Supporting Information Figure S13. C: Comparison of charge decoration in a set of protein structures with very similar backbone topologies. The indicated phylogenetic tree was generated using Euclidean distances between the 3DZDs. The numbers in black are the computed distance of corresponding branches and the branch lengths are proportional to the distances between the proteins. The PDB identifiers and DALI Z scores are 1q6y, E. coli YfdW, CoA complex (Z = 49.2); 1p5r, O. formigenes FRC, CoA complex (Z = 46.6); 1xk6 and 1xk7, E. coli CaiB in two crystal forms (Z = 34.2 and 33.8, respectively); 1x74, Mycobacterium tuberculosis α-methyl-CoA racemase (Z = 29.5); and 2g04, M. tuberculosis fatty acid-CoA racemase (Z = 28.7). The thumbnail figures are rotated to show the positively-charged (acyl-)CoA binding pocket.