A conserved motif flags acyl carrier proteins for β-branching in polyketide synthesis

Type I PKSs often utilise programmed β-branching, via enzymes of an “HMG-CoA synthase (HCS) cassette”, to incorporate various side chains at the second carbon from the terminal carboxylic acid of growing polyketide backbones. We identified a strong sequence motif in Acyl Carrier Proteins (ACPs) where β-branching is known. Substituting ACPs confirmed a correlation of ACP type with β-branching specificity. While these ACPs often occur in tandem, NMR analysis of tandem β-branching ACPs indicated no ACP-ACP synergistic effects and revealed that the conserved sequence motif forms an internal core rather than an exposed patch. Modelling and mutagenesis identified ACP Helix III as a probable anchor point of the ACP-HCS complex whose position is determined by the core. Mutating the core affects ACP functionality while ACP-HCS interface substitutions modulate system specificity. Our method for predicting β-carbon branching expands the potential for engineering novel polyketides and lays a basis for determining specificity rules

Aminoterminal amphipathic α-helix AH1 of hepatitis C virus nonstructural protein 4B possesses a dual role in RNA replication and virus production.

Nonstructural protein 4B (NS4B) is a key organizer of hepatitis C virus (HCV) replication complex formation. In concert with other nonstructural proteins, it induces a specific membrane rearrangement, designated as membranous web, which serves as a scaffold for the HCV replicase. The N-terminal part of NS4B comprises a predicted and a structurally resolved amphipathic α-helix, designated as AH1 and AH2, respectively. Here, we report a detailed structure-function analysis of NS4B AH1. Circular dichroism and nuclear magnetic resonance structural analyses revealed that AH1 folds into an amphipathic α-helix extending from NS4B amino acid 4 to 32, with positively charged residues flanking the helix. These residues are conserved among hepaciviruses. Mutagenesis and selection of pseudorevertants revealed an important role of these residues in RNA replication by affecting the biogenesis of double-membrane vesicles making up the membranous web. Moreover, alanine substitution of conserved acidic residues on the hydrophilic side of the helix reduced infectivity without significantly affecting RNA replication, indicating that AH1 is also involved in virus production. Selective membrane permeabilization and immunofluorescence microscopy analyses of a functional replicon harboring an epitope tag between NS4B AH1 and AH2 revealed a dual membrane topology of the N-terminal part of NS4B during HCV RNA replication. Luminal translocation was unaffected by the mutations introduced into AH1, but was abrogated by mutations introduced into AH2. In conclusion, our study reports the three-dimensional structure of AH1 from HCV NS4B, and highlights the importance of positively charged amino acid residues flanking this amphipathic α-helix in membranous web formation and RNA replication. In addition, we demonstrate that AH1 possesses a dual role in RNA replication and virus production, potentially governed by different topologies of the N-terminal part of NS4B

The recent development of a sweet-tasting brazzein and its potential industrial applications

Brazzein is a small heat- and pH-stable sweet-tasting protein isolated from the West African plant, Pentadiplandra brazzeana. Brazzein combines a highly sweet potency, a long history of human consumption, and a remarkable stability, giving it great potential as a natural sweetener. Due to the difficulties of obtaining brazzein from its natural source, several efforts have been made to express brazzein using various heterologous expression systems. This chapter describes the biochemical, structural, sensory, and physiological properties of brazzein. We will summarize the current knowledge of the structure-activity relationship of brazzein. The biotechnological production of brazzein using various expression systems will also be reviewed. Furthermore, the emerging application of brazzein in the food industry to replace traditional sugars by acting as a natural, good, low-calorie sweetener will be discussed