Escherichia coli FabG 3-ketoacyl-ACP reductase proteins lacking the assigned catalytic triad residues are active enzymes

Zhe Hu,Jincheng Ma,Yicai Chen,Wenhua Tong,Lei Zhu,Haihong Wang,John E Cronan

doi:10.1016/j.jbc.2021.100365

Abstract

The FabG 3-ketoacyl-acyl carrier protein (ACP) reductase of Escherichia coli has long been thought to be a classical member of the short-chain alcohol dehydrogenase/reductase (SDR) family. FabG catalyzes the essential 3-ketoacyl-ACP reduction step in the FAS II fatty acid synthesis pathway. Site-directed mutagenesis studies of several other SDR enzymes has identified three highly conserved amino acid residues, Ser, Tyr, and Lys, as the catalytic triad. Structural analyses of E. coli FabG suggested the triad S138-Y151-K155 to form a catalytically competent active site. To test this hypothesis, we constructed a series of E. coli FabG mutants and tested their 3-ketoacyl-ACP reductase activities both in vivo and in vitro. Our data show that plasmid-borne FabG mutants, including the double and triple mutants, restored growth of E. coli and Salmonella enterica fabG temperature-sensitive mutant strains under nonpermissive conditions. In vitro assays demonstrated that all of the purified FabG mutant proteins maintained fatty acid synthetic ability, although the activities of the single mutant proteins were 20% to 50% lower than that of wildtype FabG. The S138A, Y151F, and K155A residue substitutions were confirmed by tandem mass spectral sequencing of peptides that spanned all three residues. We conclude that FabG is not a classical short-chain alcohol dehydrogenase/reductase, suggesting that an alternative mode of 3-ketoacyl-ACP reduction awaits discovery.

Highlights

The architecture of fatty acid synthase (FAS) has three forms [1,2,3]
We report that substitution of the putative E. coli FabG residues S138, Y151, and K155 with A, F, or A resulted in proteins that retained FabG activity both in vivo and in vitro
To test whether the S138-Y151-K155 motif of E. coli FabG is the catalytic triad of the enzyme, panels of E. coli fabG mutants were constructed on plasmids of high copy and low copy numbers

Summary

H O Ser138 O

FabG catalytic mechanism [15]. ACP, acyl carrier protein. Price and colleagues [8, 15] postulated that E. coli FabG has a catalytic mechanism similar to that of other SDR enzymes, they observed that cofactor binding caused a significant conformational change in FabG that buried the signature lysine residue (K155). They proposed that, in E. coli FabG, protons are shuttled via a water network positioned to serve as a proton wire to the tyrosine via K155 and the 2’-hydroxyl of the nicotinamide ribose [8, 15] (Fig. 1C). We report that substitution of the putative E. coli FabG residues S138, Y151, and K155 with A, F, or A resulted in proteins that retained FabG activity both in vivo and in vitro

Results

Discussion

Experimental procedures