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The Calvin-Benson-Bassham cycle is responsible for carbon dioxide fixation in all plants, algae, and cyanobacteria. The enzyme that catalyzes the carbon dioxide-fixing reaction is ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco). Rubisco from a hyperthermophilic archaeon Thermococcus kodakarensis (Tk-Rubisco) belongs to the type III group, and shows high activity at high tempera- tures. We have previously found that replacement of the entire α-helix 6 of Tk-Rubisco with the corresponding region of the spinach enzyme (SP6 mutant) results in an improvement of catalytic performance at mesophilic tempera- tures, both in vivo and in vitro, whereas the former and latter half-replacements of the a-helix 6 (SP4 and SP5 mu- tants) do not yield such improvement. We report here the crystal structures of the wild-type Tk-Rubisco and the mutants SP4 and SP6, and discuss the relationships between their structures and enzymatic activities. A comparison among these structures shows the movement and the increase of temperature factors of α-helix 6 induced by four essential factors. We thus supposed that an increase in the flexibility of the α-helix 6 and loop 6 regions was important to increase the catalytic activ- ity of Tk-Rubisco at ambient temperatures. Based on this structural information, we constructed a new mutant, SP5-V330T, which was designed to have significantly greater flexibility in the above region, and it proved to exhibit the highest activity among all mutants examined to date. The thermostability of the SP5- V330T mutant was lower than that of wild-type Tk-Rubisco, providing further support on the relationship between flexibility and activity at ambient temperatures. [ABSTRACT FROM AUTHOR]

Uridine-diphospho-N-acetylglucosamine (UDP-GIcNAc) is a precursor of the bacterial and fungal cell wall. It is also used in a component of N-linked glycosylation and the glycosyiphosphoinositol anchor of eukaryotic proteins. It is synthesized from N-acetylglucosamine-1-phosphate (GlcNAc-1-P) and uridine-5′-triphosphate (UTP) by UDP-GlcNAc pyrophosphorylase (UAP). This is an SN2 reaction; the non-esterified oxygen atom of the GlcNAc-1-P phosphate group attacks the a-phosphate group of UTP. We determined crystal structures of UAP from Candida albicans (CaUAP1) without any ligands and also complexed with its substrate or with its product. The series of structures in different forms shows the induced fit movements of CaUAP1. Three loops approaching the ligand molecule close the active site when ligand is bound. In addition, Lys-421, instead of the metal ion in prokaryotic UAPs, is coordinated by both phosphate groups of UDP-Glc-NAc and acts as a cofactor. However, a magnesium ion enhances the enzymatic activity of CaUAP1, and thus we propose that the magnesium ion increases the affinity between UTP and the enzyme by coordinating to the α- and γ-phosphate group of UTP. [ABSTRACT FROM AUTHOR]

N-Acetylglucosamine-phosphate mutase (AGM1) is an essential enzyme in the synthetic process of UDP-N-acetylglucosamine (UDP-GlcNAc). UDP-GlcNAc is a UDP sugar that serves as a biosynthetic precursor of glycoproteins, mucopolysaccharides, and the cell wall of bacteria. Thus, a specific inhibitor of AGM1 from pathogenetic fungi could be a new candidate for an antifungal reagent that inhibits cell wall synthesis. AGM1 catalyzes the conversion of N-acetylglucosamine 6-phosphate (GlcNAc-6-P) into N-acetylglucosamine 1-phosphate (GlcNAc-1-P). This enzyme is a member of the α-D-phosphohexomutase superfamily, which catalyzes the intramolecular phosphoryl transfer of sugar substrates. Here we report the crystal structures of AGM1 from Candida albicans for the first time, both in the apoform and in the complex forms with the substrate and the product, and discuss its catalytic mechanism. The structure of AGM1 consists of four domains, of which three domains have essentially the same fold. The overall structure is similar to those of phosphohexomutases; however, there are two additional β-strands in domain 4, and a circular permutation occurs in domain 1. The catalytic cleft is formed by four loops from each domain. The N-acetyl group of the substrate is recognized by Val-370 and Asn-389 in domain 3, from which the substrate specificity arises. By comparing the substrate and product complexes, it is suggested that the substrate rotates about 180° on the axis linking C-4 and the midpoint of the C-5—O-5 bond in the reaction. [ABSTRACT FROM AUTHOR]