Insights into Acinetobacter baumannii fatty acid synthesis 3-oxoacyl-ACP reductases

Abstract

Treatments for ‘superbug’ infections are the focus for innovative research, as drug resistance threatens human health and medical practices globally. In particular, Acinetobacter baumannii (Ab) infections are repeatedly reported as difficult to treat due to increasing antibiotic resistance. Therefore, there is increasing need to identify novel targets in the development of different antimicrobials. Of particular interest is fatty acid synthesis, vital for the formation of phospholipids, lipopolysaccharides/lipooligosaccharides, and lipoproteins of Gram-negative envelopes. The bacterial type II fatty acid synthesis (FASII) pathway is an attractive target for the development of inhibitors and is particularly favourable due to the differences from mammalian type I fatty acid synthesis. Discrete enzymes in this pathway include two reductase enzymes: 3-oxoacyl-acyl carrier protein (ACP) reductase (FabG) and enoyl-ACP reductase (FabI). Here, we investigate annotated FabG homologs, finding a low-molecular weight 3-oxoacyl-ACP reductase, as the most likely FASII FabG candidate, and high-molecular weight 3-oxoacyl-ACP reductase (HMwFabG), showing differences in structure and coenzyme preference. To date, this is the second bacterial high-molecular weight FabG structurally characterized, following FabG4 from Mycobacterium. We show that ΔAbHMwfabG is impaired for growth in nutrient rich media and pellicle formation. We also modelled a third 3-oxoacyl-ACP reductase, which we annotated as AbSDR. Despite containing residues for catalysis and the ACP coordinating motif, biochemical analyses showed limited activity against an acetoacetyl-CoA substrate in vitro. Inhibitors designed to target FabG proteins and thus prevent fatty acid synthesis may provide a platform for use against multidrug-resistant pathogens including A. baumannii.

Structural and functional characterization of FabG4 from Mycolicibacterium smegmatis

“…The estimated molecular weight is roughly double the predicted monomer weight, indicating that it forms a dimer. The SEC data support the PISA analysis; additionally, both of the other characterized high-molecularweight ketoacyl reductases, M. tuberculosis FabG4 and A. baumannii HMwFabG, are dimeric (Dutta et al, 2011;Cross et al, 2021).…”

“…MsFabG4 shares a similar overall structure with the two other solved structures from M. tuberculosis and A. baumannii, which also contain a flavodoxin-type domain followed by a oxidoreductase domain (Fig. 1e; Dutta et al, 2011;Cross et al, 2021). PDBeFold analysis was performed to identify other similar structures to MsFabG4 (Krissinel & Henrick, 2004).…”

“…Only one other HMwFabG has been characterized, Actinobacter baumannii HMwFabG (AbHMw-FabG), and it also preferentially uses NADH for reductase activity (Cross et al, 2021). Structural characterization of MtFabG4 and AbHMwFabG revealed two distinct domains: an N-terminal 'flavodoxin-type' domain and a C-terminal oxidoreductase domain (Dutta et al, 2011(Dutta et al, , 2013Cross et al, 2021). This N-terminal domain is absent in LMwFabGs, while the C-terminal domain resembles the prototypical LMwFabG structure, like that of FabG1/MabA (Dutta et al, 2011;Cross et al, 2021).…”

“…FabG4 is also found in the cell-envelope membrane fraction of M. tuberculosis and is hypothesized to be involved in mycobacterial biofilm formation (Sharma et al, 2019(Sharma et al, , 2022. In A. baumannii, HMwFabG was found to be essential in several processes, including motility, pellicle formation and growth in nutrient-rich broth/conditions (Cross et al, 2021). Additionally, treatment with antibiotics led to downregulation of the low-molecular-weight FabG and upregulation of HMwFabG in A. baumannii, suggesting that a switch between FabG homologs may be beneficial to survival (Henry et al, 2015).…”

Structural Characterization of Porcine Adeno-Associated Virus Capsid Protein with Nuclear Trafficking Protein Importin Alpha Reveals a Bipartite Nuclear Localization Signal

“…The IMPα2:AAV Po1 Cap-BR complex was successfully crystallized [ Figure 2 D] via the hanging drop vapour diffusion method [ 61 ], and short rod-shaped crystals diffracted [ Table 1 ] on the MX2 beamline at the Australian Synchrotron. The data were indexed and integrated in iMOSFLM [ 53 ], prior to merging and scaling performed in Aimless [ Table 1 ].…”