Biochemical analysis of a sugar 4,6‐dehydratase from Acanthamoeba polyphaga Mimivirus

Abstract

The exciting discovery of the giant DNA Mimivirus in 2003 challenged the conventional description of viruses in a radical way, and since then, dozens of additional giant viruses have been identified. It has now been demonstrated that the Mimivirus genome encodes for the two enzymes required for the production of the unusual sugar 4-amino-4,6-dideoxy-D-glucose, namely a 4,6-dehydratase and an aminotransferase. In light of our long-standing interest in the bacterial 4,6-dehydratases and in unusual sugars in general, we conducted a combined structural and functional analysis of the Mimivirus 4,6-dehydratase referred to as R141. For this investigation, the threedimensional X-ray structure of R141 was determined to 2.05 Å resolution and refined to an R-factor of 18.3%. The overall fold of R141 places it into the short-chain dehydrogenase/reductase (SDR) superfamily of proteins. Whereas its molecular architecture is similar to that observed for the bacterial 4,6-dehydratases, there are two key regions where the polypeptide chain adopts different conformations. In particular, the conserved tyrosine that has been implicated as a catalytic acid or base in SDR superfamily members is splayed away from the active site by nearly 12 Å, thereby suggesting that a major conformational change must occur upon substrate binding. In addition to the structural analysis, the kinetic parameters for R141 using either dTDP-D-glucose or UDP-D-glucose as substrates were determined. Contrary to a previous report, R141 demonstrates nearly identical catalytic efficiency with either nucleotide-linked sugar. The data presented herein represent the first threedimensional model for a viral 4,6-dehydratase and thus expands our understanding of these fascinating enzymes. K E Y W O R D SAcanthamoeba polyphaga Mimivirus, dideoxysugar, giant viruses, UDP-D-glucose 4,6-dehydratase, UDP-D-viosamine, viral glycans, X-ray structure Abbreviations: dTDP, thymidine diphosphate; ESI, electrospray ionization; HEPES, N-2-hydroxyethylpiperazine-N 0 -2-ethanesulfonic acid; HEPPS, N-2-hydroxyethylpiperazine-N 0 -3-propanesulfonic acid; HPLC, high-performance liquid chromatography; NAD + , nicotinamide adenine dinucleotide (oxidized); NADH, nicotinamide adenine dinucleotide (reduced); NADP + , nicotinamide adenine dinucleotide phosphate (oxidized); NADPH, nicotinamide adenine dinucleotide phosphate (reduced); Ni-NTA, nickel-nitrilotriacetic acid; PCR, polymerase chain reaction; PLP, pyridoxal 5 0 -phosphate; Qui4N, 4-amino-4,6-dideoxy-D-glucose; TEV, Tobacco Etch Virus; Tris, tris-(hydroxymethyl)aminomethane; UDP, uridine diphosphate.

The high‐resolution structure of a UDP‐L‐rhamnose synthase from Acanthamoeba polyphagaMimivirus

“…The electron density for the dinucleotide shows that the NADP + had been hydrolyzed to ADP‐ribose 2′‐phosphate. This phenomenon has been previously observed in several of our structural investigations on dinucleotide‐dependent enzymes, and it is most likely an artifact of the experiment 17,20 . The electron density for the dinucleotide in subunit 2 also indicated that the nicotinamide ring had been hydrolyzed from the cofactor.…”

“…16 We recently reported the X-ray structure of R141 from Mimivirus to 2.05 Å resolution. 17 Our results demonstrated several significant architectural differences between the Mimivirus 4,6-dehydratase and those previously characterized bacterial enzymes. As a continuation of our studies on sugar-modifying enzymes from Mimivirus, herein we report a structural and enzymological characterization of L780.…”

“…We recently reported the X‐ray structure of R141 from Mimivirus to 2.05 Å resolution 17 . Our results demonstrated several significant architectural differences between the Mimivirus 4,6‐dehydratase and those previously characterized bacterial enzymes.…”

“…A solution of 2.5 mM UDP‐ d ‐glucose, 3.0 mM NADPH, and 50 mM HEPPS was adjusted to pH 8.0. The R141 dehydratase, prepared as previously described, 17 was added to a final concentration of 0.5 mg/ml and L780 added to a final concentration of 1 mg/ml. The reaction was allowed to proceed overnight at room temperature.…”

“…This phenomenon has been previously observed in several of our structural investigations on dinucleotide-dependent enzymes, and it is most likely an artifact of the experiment. 17,20 The electron density for the dinucleotide in subunit 2 also indicated that the nicotinamide ring had been hydrolyzed from the cofactor. The electron density corresponding to UDP-Lrhamnose was unambiguous in both subunits, however ( Figure 2).…”