Malonyl-acyl carrier protein decarboxylase activity promotes fatty acid and cell envelope biosynthesis in Proteobacteria

Abstract

Bacterial fatty acid synthesis in Escherichia coli is initiated by the condensation of an acetyl-CoA with a malonyl-acyl carrier protein (ACP) by the β-ketoacyl-ACP synthase III enzyme, FabH. E. coli Δ fabH knockout strains are viable because of the yiiD gene that allows FabH-independent fatty acid synthesis initiation. However, the molecular function of the yiiD gene product is not known. Here, we show the yiiD gene product is a m alonyl- A CP d ecarboxylase (MadA). MadA has two independently folded domains: an amino-terminal N -acetyl transferase (GNAT) domain (MadA N ) and a carboxy-terminal hot dog dimerization domain (MadA C ) that encodes the malonyl-ACP decarboxylase function. Members of the proteobacterial Mad protein family are either two domain MadA (GNAT-hot dog) or standalone MadB (hot dog) decarboxylases. Using structure-guided, site-directed mutagenesis of MadB from Shewanella oneidensis , we identified Asn45 on a conserved catalytic loop as critical for decarboxylase activity. We also found that MadA, MadA C , or MadB expression all restored normal cell size and growth rates to an E. coli Δ fabH strain, whereas the expression of MadA N did not. Finally, we verified that GlmU, a bifunctional glucosamine-1-phosphate N -acetyl transferase/ N -acetyl-glucosamine-1-phosphate uridylyltransferase that synthesizes the key intermediate UDP-GlcNAc, is an ACP binding protein. Acetyl-ACP is the preferred glucosamine-1-phosphate N -acetyl transferase/ N -acetyl-glucosamine-1-phosphate uridylyltransferase substrate, in addition to being the substrate for the elongation-condensing enzymes FabB and FabF. Thus, we conclude that the Mad family of malonyl-ACP decarboxylases supplies acetyl-ACP to support the initiation of fatty acid, lipopolysaccharide, peptidoglycan, and enterobacterial common antigen biosynthesis in Proteobacteria.

A statistical genomics framework to trace bacterial genomic predictors of clinical outcomes in Staphylococcus aureus bacteraemia

“…Again, some of these genes (such as walK and vraG in cohort B) are known to be associated with vancomycin resistance but were not identified in the mutations analysis, highlighting the potential of the gene-burden test to increase the power of GWAS. A strong association was found with the SAUSA300_0956 locus, a GNAT-family acetyltransferase (Majorek, Osinski et al 2017, Whaley, Radka et al 2021). The function of the protein in S. aureus is not well understood, however, some indirect evidence may point to a role in cell-wall synthesis or turn-over.…”

“…First, it is located between the autolysin atl and the methicillin resistance factor fmtA (Qamar and Golemi-Kotra 2012) . Second, an E. coli homolog of SAUSA300_0956 was linked to membrane biosynthesis (Whaley, Radka et al 2021). Another interest association involved the SAUSA300_1220 locus.…”

“…We found two significant associations in cohort C. These involved truncations in metN2 , a methionine transporter, and protein-modifying mutations in plsX . The latter gene is a phosphate acyltransferase and is part of the phospholipid synthesis pathway (Whaley, Radka et al 2021). It interacts with the fatty-acid kinase fakA which has been linked to virulence factors expression (Parsons, Broussard et al 2014) and resistance to antimicrobial peptides (Li, Rigby et al 2009).…”

Loss of β-ketoacyl acyl carrier protein synthase III activity restores multidrug-resistantEscherichia colisensitivity to previously ineffective antibiotics

“…However, this essentiality is bypassed by the product of the yiiD gene (renamed MadA) (49). The bypass mechanism involves the decarboxylation of malonyl ACP to produce acetyl ACP (50). Unlike acetyl CoA, which can only be integrated into the initiation step of FA biosynthesis by FabH, this substrate can be used by FabB/FabF to bypass the loss of KAS III activity in E. coli (50).…”

“…The bypass mechanism involves the decarboxylation of malonyl ACP to produce acetyl ACP (50). Unlike acetyl CoA, which can only be integrated into the initiation step of FA biosynthesis by FabH, this substrate can be used by FabB/FabF to bypass the loss of KAS III activity in E. coli (50). However, the supply of FA to integrate into membrane biogenesis by the MadA bypass is highly restrictive, and as such, earlier E. coli K-12 studies reported that Δ fabH cells are tiny (26, 27).…”