The genome of a Bacteroidetes inhabitant of the human gut encodes a structurally distinct enoyl-acyl carrier protein reductase (FabI)

Abstract

Enoyl-acyl carrier protein reductase (FabI) catalyzes a rate-controlling step in bacterial fatty-acid synthesis and is a target for antibacterial drug development. A phylogenetic analysis shows that FabIs fall into four divergent clades. Members of clades 1–3 have been structurally and biochemically characterized, but the fourth clade, found in members of phylum Bacteroidetes, is uncharacterized. Here, we identified the unique structure and conformational changes that distinguish clade 4 FabIs. Alistipes finegoldii is a prototypical Bacteroidetes inhabitant of the gut microbiome. We found that A. finegoldii FabI (AfFabI) displays cooperative kinetics and uses NADH as a cofactor, and its crystal structure at 1.72 Å resolution showed that it adopts a Rossmann fold as do other characterized FabIs. It also disclosed a carboxyl-terminal extension that forms a helix–helix interaction that links the protomers as a unique feature of AfFabI. An AfFabI·NADH crystal structure at 1.86 Å resolution revealed that this feature undergoes a large conformational change to participate in covering the NADH-binding pocket and establishing the water channels that connect the active site to the central water well. Progressive deletion of these interactions led to catalytically compromised proteins that fail to bind NADH. This unique conformational change imparted a distinct shape to the AfFabI active site that renders it refractory to a FabI drug that targets clade 1 and 3 pathogens. We conclude that the clade 4 FabI, found in the Bacteroidetes inhabitants of the gut, have several structural features and conformational transitions that distinguish them from other bacterial FabIs.

Do Go Chasing Waterfalls: Enoyl Reductase (FabI) in Complex with Inhibitors Stabilizes the Tetrameric Structure and Opens Water Channels

“…Further, PCA extreme motions suggest a larger amplitude of movement in the C-terminus, in comparison to other substructures. Interestingly, recent findings using truncated mutants at the C-terminus suggest the loss of interactions with other subunits in the tetramer. In our simulations, the C-terminus interacts with the L:146–154 loop of another monomer (see the Supporting Information, Table S1 and associated discussion), displaying an additional indirect effect on the monomer’s conformational changes (see Figure ).…”

“…Those binding modes rely not only upon direct interaction with binding site residues but also on promoting a network of intramolecular interactions that affects tetramer interfaces (clearly the QR interface but also others), suggesting cooperativity among FabI’s subunits. Last but not least, the observed protein motions and their related mechanistic implications in addition to experimental data , could open a brand new possibility to target allosteric sites, aiming to increase the specificity and diversity of novel inhibitors. , …”

Biochemical characterization of the first step in sulfonolipid biosynthesis in Alistipes finegoldii

“…A. finegoldii genes alfi_1465 (Uniprot ID: I3YLE0) and alfi_1224 (Uniprot ID: I3YKQ4) DNA sequences were optimized for gene expression in E. coli using GeneArt Gene Synthesis Technology (Life Technologies) and synthesized with 5′-NdeI and 3′-EcoRI restriction sites for cloning into the pPJ131 vector for constitutive expression in E. coli ( 48 ). For protein purification, the alfi_1224 construct was cloned into the pET28b plasmid to make pET-SulA using the same restriction sites to introduce an amino-terminal His 6 -tag.…”

“…Samples were added to the wells of ThermoGrid optically clear PCR places (Denville Scientific) and Sypro Orange Dye was added to a final concentration of 2.5×. The plates were centrifuged at 1000 g for 5 min and then analyzed by the ABI 7300 real-time PCR system as described previously ( 48 ). Each sample was replicated three times, and the thermal melting point of each replicate was determined independently by fitting the data points to the Boltzmann equation using Graph-Pad software.…”

Sunflower (Helianthus annuus) fatty acid synthase complex: β-Ketoacyl-[acyl carrier protein] reductase genes

“…These observations resulted in denotation of KARs as enzymes with a catalytic tetrad (Filling et al, 2002). Recent characterization of two FABI enoyl reductases also belonging to the SDR group using the NADPH reductant were denoted as having a catalytic dyad (Tyr and Lys) with other residues carrying out analogous functions of the KARs Ser and Asn (Radka et al, 2020;Rao et al, 2020). While future work is needed to determine whether KARs have a dyad instead of a tetrad catalytic site, we use the tetrad designation herein.…”