Structure and function of an N‐acetyltransferase from the human pathogen Acinetobacter baumannii isolate BAL_212

Abstract

Acinetobacter baumannii is a Gram-negative bacterium commonly found in soil and water that can cause human infections of the blood, lungs, and urinary tract. Of particular concern is its prevalence in health-care settings where it can survive on surfaces and shared equipment for extended periods of time. The capsular polysaccharide surrounding the organism is known to be the major contributor to virulence. The structure of the K57 capsular polysaccharide produced by A. baumannii isolate BAL_212 from Vietnam was recently shown to contain the rare sugar 4-acetamido-4,6-dideoxy-D-glucose. Three enzymes are required for its biosynthesis, one of which is encoded by the gene H6W49_RS17300 and referred to as VioB, a putative N-acetyltransferase. Here, we describe a combined structural and functional analysis of VioB. Kinetic analyses show that the enzyme does, indeed, function on dTDP-4-amino-4,6-dideoxy-D-glucose with a catalytic efficiency of 3.9 x 10 4 M À1 s À1 (±6000), albeit at a reduced value compared to similar enzymes. Three high-resolution X-ray structures of various enzyme/ligand complexes were determined to resolutions of 1.65 Å or better. One of these models represents an intermediate analogue of the tetrahedral transition state. Differences between the VioB structure and those determined for the N-acetyltransferases from Campylobacter jejuni (PglD), Caulobacter crescentus (PerB), and Psychrobacter cryohalolentis (Pcryo_0637) are highlighted. Taken together, this investigation sheds new insight into the Type I sugar N-acetyltransferases. K E Y W O R D S 4,6-dideoxyhexoses, 4-acetamido-4,6-dideoxy-D-glucose, 4-amino-4,6-dideoxy-D-glucose, Acinetobacter baumannii, carbohydrate, enzyme structure, N-acetyltransferase 1 | INTRODUCTIONAcinetobacter baumannii is an opportunistic Gram-negative bacterium that has recently emerged as a significant human health threat worldwide. 1,2 Due to the increasing rise of A. baumannii infections in hospital settings, and its extensive antibiotic resistance spectrum, it has been designated as

Investigation of the enzymes required for the biosynthesis of 2,3‐diacetamido‐2,3‐dideoxy‐d‐glucuronic acid in Psychrobacter cryohalolentisK5T

“…Shown in Figure 4 is the initial velocity versus UDP‐GlcNAc3NA concentration for Pcryo_O615. The enzyme demonstrates a catalytic efficiency of 1.1 (±0.2) × 10 4 M −1 s −1 for UDP‐GlcNAc3NA, which is similar to that observed for the N ‐acetyltransferase from the human pathogen A. baumannii isolate BAL‐212 (3.9 × 10 4 M −1 s −1 ) 16 …”

“…The crystals utilized belonged to the P 2 1 space group with six subunits in the asymmetric unit. Previous investigations have shown that the nucleotide‐linked sugar N ‐acetyltransferases function as trimers with C 3 symmetry 16,19–26 . A ribbon representation of one of the trimers is presented in Figure 7a and as can be seen, the active sites are wedged between the subunits.…”

“…The enzyme demonstrates a catalytic efficiency of 1.1 (±0.2) Â 10 4 M À1 s À1 for UDP-GlcNAc3NA, which is similar to that observed for the Nacetyltransferase from the human pathogen A. baumannii isolate BAL-212 (3.9 Â 10 4 M À1 s À1 ). 16 All kinetic experiments were performed utilizing tagfree forms of the enzymes. Relevant kinetic parameters are provided in Table 1.…”

“…Previous investigations have shown that the nucleotide-linked sugar N-acetyltransferases function as trimers with C3 symmetry. 16,[19][20][21][22][23][24][25][26] A ribbon representation of one of the trimers is presented in Figure 7a and as can be seen, the active sites are wedged between the subunits. Each subunit assumes a lefthanded β-helix motif composed of 22 β-strands, which is followed by two α-helices delineated by Asp 162-Ser 168 and Glu 172-Arg 186.…”