Diversity of membrane transport proteins for vitamins in bacteria and archaea

BACKGROUND: All organisms use cofactors to extend the catalytic capacities of proteins. Many bacteria and archaea can synthesize cofactors from primary metabolites, but there are also prokaryotes that do not have the complete biosynthetic pathways for all essential cofactors. These organisms are dependent on the uptake of cofactors, or at least their precursors that cannot be synthesized, from the environment. Even in those organisms that contain complete biosynthetic pathways membrane transporters are usually present, because the synthesis of cofactors is more costly than uptake.SCOPE OF REVIEW: Here we give an overview of bacterial and archaeal transport systems for B-type vitamins, which are either cofactors or precursors thereof.MAJOR CONCLUSIONS: Prokaryotic vitamin transporters are extremely diverse, and found in many families of transporters. A few of these transport systems have been characterized in detail, but for most of them mechanistic insight is lacking.GENERAL SIGNIFICANCE: The lack of structural and functional understanding of bacterial vitamin transporters is unfortunate because they may be targets for new antibiotics. This article is part of a Special Issue entitled Structural biochemistry and biophysics of membrane proteins. Guest Editor: Bjorn Pedersen.</p

Structural and Functional Characterization of NadR from Lactococcus lactis

NadR is a bifunctional enzyme that converts nicotinamide riboside (NR) into nicotinamide mononucleotide (NMN), which is then converted into nicotinamide adenine dinucleotide (NAD). Although a crystal structure of the enzyme from the Gram-negative bacterium Haemophilus influenzae is known, structural understanding of its catalytic mechanism remains unclear. Here, we purified the NadR enzyme from Lactococcus lactis and established an assay to determine the combined activity of this bifunctional enzyme. The conversion of NR into NAD showed hyperbolic dependence on the NR concentration, but sigmoidal dependence on the ATP concentration. The apparent cooperativity for ATP may be explained because both reactions catalyzed by the bifunctional enzyme (phosphorylation of NR and adenylation of NMN) require ATP. The conversion of NMN into NAD followed simple Michaelis-Menten kinetics for NMN, but again with the sigmoidal dependence on the ATP concentration. In this case, the apparent cooperativity is unexpected since only a single ATP is used in the NMN adenylyltransferase catalyzed reaction. To determine the possible structural determinants of such cooperativity, we solved the crystal structure of NadR from L. lactis (NadRLl). Co-crystallization with NAD, NR, NMN, ATP, and AMP-PNP revealed a ‘sink’ for adenine nucleotides in a location between two domains. This sink could be a regulatory site, or it may facilitate the channeling of substrates between the two domains

Structure, function, evolution, and application of bacterial Pnu-type vitamin transporters

Many bacteria can take up vitamins from the environment via specific transport machineries. Uptake is essential for organisms that lack complete vitamin biosynthesis pathways, but even in the presence of biosynthesis routes uptake is likely preferred, because it is energetically less costly. Pnu transporters represent a class of membrane transporters for a diverse set of B-type vitamins. They were identified 30 years ago and catalyze transport by the mechanism of facilitated diffusion, without direct coupling to ATP hydrolysis or transport of coupling ions. Instead, directionality is achieved by metabolic trapping, in which the vitamin substrate is converted into a derivative that cannot be transported, for instance by phosphorylation. The recent crystal structure of the nicotinamide riboside transporter PnuC has provided the first insights in substrate recognition and selectivity. Here, we will summarize the current knowledge about the function, structure, and evolution of Pnu transporters. Additionally, we will highlight their role for potential biotechnological and pharmaceutical applications

The twisted relation between Pnu and SWEET transporters

The evolutionary relation between sugar and vitamin transporters from the SWEET and Pnu families is un- clear. They have similar 3D structures, but differ in the topology of their secondary structure elements, and lack significant sequence similarity. Here we analyze the structures and sequences of different members of the SWEET and Pnu transporter families and propose an evolutionary pathway by which they may have diverged from a common ancestor. A 3D domain swapping event can explain the topological differences between the families, as well as the puzzling observation that a highly conserved and essential sequence motif of the SWEET family (the PQ loop) is absent from the Pnu transporter

Crystal structure of the vitamin B3 transporter PnuC, a full-length SWEET homolog

PnuC transporters catalyze cellular uptake of the NAD(+) precursor nicotinamide riboside (NR) and belong to a large superfamily that includes the SWEET sugar transporters. We present a crystal structure of Neisseria mucosa PnuC, which adopts a highly symmetrical fold with 3 + 1 + 3 membrane topology not previously observed in any protein. The high symmetry of PnuC with a single NR bound in the center suggests a simple alternating-access translocation mechanism