Investigation of the enzymes required for the biosynthesis of 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid in Psychrobacter cryohalolentis K5T.

Psychrobacter cryohalolentis K5T is a Gram-negative bacterium first isolated from Siberian permafrost in 2006. It has a complex O-antigen containing l-rhamnose, d-galactose, two diacetamido-sugars, and one triacetamido-sugar. The biosynthetic pathway for one of the diacetamido-sugars, namely 2,3-diacetamido-2,3-dideoxy-d-glucuronic acid, is presently unknown. Utilizing the published genome sequence of P. cryohalolentis K5T , we hypothesized that the genes designated Pcryo_0613, Pcryo_0614, Pcryo_0616, and Pcryo_0615 encode for a uridine dinucleotide (UDP)-N-acetyl-d-glucosamine 6-dehydrogenase, an nicotinamide adenine dinucleotide (oxidized) (NAD+ )-dependent dehydrogenase, a pyridoxal 5'-phosphate (PLP)-dependent aminotransferase, and an N-acetyltransferase, respectively, activities of which would be required for the biosynthesis of this unusual carbohydrate. Here we present the cloning, overexpression, and purification of these hypothetical proteins. Kinetic data on the enzymes encoded by Pcryo_0613, Pcryo_0614, and Pcryo_0615 confirmed their postulated biochemical activities. In addition, the high-resolution X-ray structures of both the internal and external aldimine forms of the aminotransferase were determined to 1.25 and 1.0 Å, respectively. Finally, the three-dimensional architecture of the N-acetyltransferase in complex with its substrate and coenzyme A was solved to 1.8 Å resolution. Strikingly, the N-acetyltransferase was shown to adopt a new motif for UDP-sugar binding. The data presented herein provide additional insight into sugar biosynthesis in Gram-negative bacteria.

Characterization of an aminotransferase from Acanthamoeba polyphaga Mimivirus.

Acanthamoeba polyphaga Mimivirus, a complex virus that infects amoeba, was first reported in 2003. It is now known that its DNA genome encodes for nearly 1,000 proteins including enzymes that are required for the biosynthesis of the unusual sugar 4-amino-4,6-dideoxy-d-glucose, also known as d-viosamine. As observed in some bacteria, the pathway for the production of this sugar initiates with a nucleotide-linked sugar, which in the Mimivirus is thought to be UDP-d-glucose. The enzyme required for the installment of the amino group at the C-4' position of the pyranosyl moiety is encoded in the Mimivirus by the L136 gene. Here, we describe a structural and functional analysis of this pyridoxal 5'-phosphate-dependent enzyme, referred to as L136. For this analysis, three high-resolution X-ray structures were determined: the wildtype enzyme/pyridoxamine 5'-phosphate/dTDP complex and the site-directed mutant variant K185A in the presence of either UDP-4-amino-4,6-dideoxy-d-glucose or dTDP-4-amino-4,6-dideoxy-d-glucose. Additionally, the kinetic parameters of the enzyme utilizing either UDP-d-glucose or dTDP-d-glucose were measured and demonstrated that L136 is efficient with both substrates. This is in sharp contrast to the structurally related DesI from Streptomyces venezuelae, whose three-dimensional architecture was previously reported by this laboratory. As determined in this investigation, DesI shows a profound preference in its catalytic efficiency for the dTDP-linked sugar substrate. This difference can be explained in part by a hydrophobic patch in DesI that is missing in L136. Notably, the structure of L136 reported here represents the first three-dimensional model for a virally encoded PLP-dependent enzyme and thus provides new information on sugar aminotransferases in general.