Structural Basis for the Stereochemical Control of Amine Installation in Nucleotide Sugar Aminotransferases

Abstract

Sugar aminotransferases (SATs) are an important class of tailoring enzymes that catalyze the 5′-pyridoxal phosphate (PLP)-dependent stereo- and regiospecific installation of an amino group from an amino acid donor (typically l-Glu or l-Gln) to a corresponding ketosugar nucleotide acceptor. Herein we report the strategic structural study of two homologous C4 SATs (Micromonospora echinospora CalS13 and Escherichia coli WecE) that utilize identical substrates but differ in their stereochemistry of aminotransfer. This study reveals for the first time a new mode of SAT sugar nucleotide binding and, in conjunction with previously reported SAT structural studies, p.rovides the basis from which to propose a universal model for SAT stereo- and regiochemical control of amine installation. Specifically, the universal model put forth highlights catalytic divergence to derive solely from distinctions within nucleotide sugar orientation upon binding within a relatively fixed SAT active site where the available ligand bound structures of the three out of four representative C3 and C4 SAT examples provide a basis for the overall model. Importantly, this study presents a new predictive model to support SAT functional annotation, biochemical study and rational engineering.

Structural characterization of AtmS13, a putative sugar aminotransferase involved in indolocarbazole AT2433 aminopentose biosynthesis

“…The structure of the biochemically characterized SAT (CalS13) involved in calicheamicin aminohexose biosynthesis was recently solved in ternary complex with PLP and TDP-4-keto-6-deoxy-a-D-glucose (PDB entry 4ZAS). 40 An overlay of the AtmS13 and CalS13 structures resulted in a root mean square deviation of 0.83 Å between the two structures. Upon analysis of the active site of AtmS13 and CalS13, the key PLP interacting residues are largely conserved, a minor exception being the PLP C5 hydroxyl hydrogen bond partner AtmS13 His161 is replaced by CalS13 Gln176 [Fig.…”

Enterobacterial Common Antigen: Synthesis and Function of an Enigmatic Molecule

“…The structure indicates a homodimeric protein; however, a previous gel filtration experiment suggested a homotetrameric conformation (124). As is common for sugar aminotransferases, the WecE active site contains a conserved lysine that binds the catalytic cofactor, 5=-pyridoxal phosphate, an aspartate important for cofactor activation, and a conserved glutamine (125).…”

“…The third reaction is catalyzed by WecE (dTDP-4-dehydro-6-deoxy- d -glucose transaminase), which converts dTDP-4-keto-6-deoxy- d -glucose to dTDP-4-amino-4,6-dideoxy-a- d -galactose (dTDP-Fuc4N) using glutamate as the amino donor ( 103 , 124 ). A WecE crystal structure has been solved at a resolution of 2.24 Å ( 125 ). The structure indicates a homodimeric protein; however, a previous gel filtration experiment suggested a homotetrameric conformation ( 124 ).…”

Structural investigation on WlaRG from Campylobacter jejuni: A sugar aminotransferase

“…21 This enzyme is involved in the biosynthesis of UDP-4-amino-4-deoxy-L-arabinose, whose addition to lipid A results in bacterial resistance to cationic antimicrobial peptides. 22 Other enzymes with known three-dimensional structures that function on C-4 0 keto sugar substrates include PseC from Helicobacter pylori, 23 DesI from Streptomyces venezuelae, 24 perosamine synthase from Caulobacter crescentus, 25 WecE from Escherichia coli, 26 and PglE from C. jejuni. 18 In contrast, only three models have been reported to date for those enzymes that function on C-3 0 keto moieties: DesV from Streptomyces venezuelae, QdtB from Thermoanaerobacterium thermosaccharolyticum E207-71, and WbpE from P. aeruginosa.…”

“…This enzyme is involved in the biosynthesis of UDP‐4‐amino‐4‐deoxy‐ l ‐arabinose, whose addition to lipid A results in bacterial resistance to cationic antimicrobial peptides . Other enzymes with known three‐dimensional structures that function on C‐4′ keto sugar substrates include PseC from Helicobacter pylori , DesI from Streptomyces venezuelae , perosamine synthase from Caulobacter crescentus , WecE from Escherichia coli, and PglE from C. jejuni …”