The molecular details that govern the specific interactions between acyl carrier protein (ACP) and the enzymes of fatty acid biosynthesis are unknown. We investigated the mechanism of ACP⅐protein interactions using a computational analysis to dock the NMR structure of ACP with the crystal structure of ␤-ketoacyl-ACP synthase III (FabH) and experimentally tested the model by the biochemical analysis of FabH mutants. The activities of the mutants were assessed using both an ACP-dependent and an ACP-independent assay. The ACP interaction surface was defined by mutations that compromised FabH activity in the ACP-dependent assay but had no effect in the ACP-independent assay. ACP docked to a positively charged/hydrophobic patch adjacent to the active site tunnel on FabH, which included a conserved arginine (Arg-249) that was required for ACP docking. Kinetic analysis and direct binding studies between FabH and ACP confirmed the identification of Arg-249 as critical for FabH⅐ACP interaction. Our experiments reveal the significance of the positively charged/ hydrophobic patch located adjacent to the active site cavities of the fatty acid biosynthesis enzymes and the high degree of sequence conservation in helix II of ACP across species.The 4Ј-phosphopantetheine prosthetic group is a central and universal feature in the mechanism of fatty acid biosynthesis that provides two crucial functionalities to the process: a long and flexible arm that can reach into active sites and a terminal sulfhydryl group for the attachment of acyl groups through a thioester linkage. Two types of fatty acid synthase architectures exist in nature, and the 4Ј-phosphopantetheine moiety operates quite differently in each type. The type I, or associated system, found in metazoans, consists of a single large polypeptide containing multiple active centers. In this system, the prosthetic group with its attached nascent fatty acid swings between the active sites in the multifunctional complex. This contrasts with the type II or dissociated system found in bacteria and plants in which the active centers reside in discrete protein molecules. Here, the 4Ј-phosphopantetheine moiety is covalently attached to acyl carrier protein (ACP), 1 a small protein that sequentially delivers the lipid intermediates to the active site of each enzyme in the pathway.ACP is a small, acidic and highly conserved protein with a molecular mass of 8847 Da (1). In Escherichia coli, it is encoded by the acpP gene that is located within a cluster of other fatty acid biosynthetic genes (2, 3). Biophysical (4) and solution NMR structural studies (5) show that the protein is an asymmetric monomer composed of three ␣-helices packed into a bundle with an extended and flexible loop at one end (6, 7). The fatty acid intermediates are attached to the terminal sulfhydryl of the 4Ј-phosphopantetheine prosthetic group, which in turn, is covalently attached to serine 36 via a phosphodiester linkage (8). In addition to fatty acid biosynthesis, ACP also supplies acyl groups for the synthesis ...

Background The ideal SARs-CoV-2 testing method would be accurate and also be patient-performed to reduce exposure to healthcare workers.  The aim of this study was to compare patient-performed testing based on a morning saliva sample with the current standard testing method, healthcare worker-collected sampling via a nasopharyngeal swab (NPS). Methods This was a prospective single center study which recruited 217 asymptomatic adult male participants in a COVID-19 quarantine center who had tested positive for SARS-CoV-2 8-10 days prior isolation. Paired NPS and saliva specimens were collected and processed within 5 hours of sample collection. Real time reverse transcriptase polymerase chain reaction (RT-PCR) targeting Envelope (E) and RNA-dependent RNA polymerase (RdRp) genes was performed and the results were compared. Results Overall, 160 of the 217 (74%) participants tested positive for Covid-19 based on saliva, NPS, or both testing methods. The detection rate for SARS-CoV-2 was higher in saliva compared to NPS testing (93.1%, 149/160 vs 52.5%, 84/160, p<0.001). The concordance between the two tests was 45.6% (virus was detected in both saliva and NPS in 73/160), while 47.5% were discordant (87/160 tested positive for one while negative for the other). The Ct values for E and RdRp genes were significantly lower in saliva specimens compared to NP swab specimens. Conclusions Our findings demonstrate that saliva is a better alternative specimen for detection of SARS-CoV-2. Taking into consideration, the simplicity of specimen collection, shortage of PPE and the transmissibility of the virus, saliva could enable self-collection for an accurate SARS-CoV-2 surveillance testing.

Crystallization of the 1:1 molecular complex between the beta-lactamase TEM-1 and the beta-lactamase inhibitory protein BLIP has provided an opportunity to put a stringent test on current protein-docking algorithms. Prior to the successful determination of the structure of the complex, nine laboratory groups were given the refined atomic coordinates of each of the native molecules. Other than the fact that BLIP is an effective inhibitor of a number of beta-lactamase enzymes (KI for TEM-1 approximately 100 pM) no other biochemical or structural data were available to assist the practitioners in their molecular docking. In addition, it was not known whether the molecules underwent conformational changes upon association or whether the inhibition was competitive or non-competitive. All six of the groups that accepted the challenge correctly predicted the general mode of association of BLIP and TEM-1.

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