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Bacteria respond to environmental changes by inducing transcription of some genes and repressing others. Sialic acids, which coat human cell surfaces, are a nutrient source for pathogenic and commensal bacteria. The Escherichia coli GntR-type transcriptional repressor, NanR, regulates sialic acid metabolism, but the mechanism is unclear. Here, we demonstrate that three NanR dimers bind a (GGTATA) 3 -repeat operator cooperatively and with high affinity. Single-particle cryo-electron microscopy structures reveal the DNA-binding domain is reorganized to engage DNA, while three dimers assemble in close proximity across the (GGTATA) 3 -repeat operator. Such an interaction allows cooperative protein-protein interactions between NanR dimers via their N-terminal extensions. The effector, N-acetylneuraminate, binds NanR and attenuates the NanR-DNA interaction. The crystal structure of NanR in complex with N-acetylneuraminate reveals a domain rearrangement upon N-acetylneuraminate binding to lock NanR in a conformation that weakens DNA binding. Our data provide a molecular basis for the regulation of bacterial sialic acid metabolism.

Pyruvate kinase catalyses the final step of the glycolytic pathway in central energy metabolism. The monomeric structure comprises three domains: a catalytic TIM-barrel, a regulatory domain involved in allosteric activation, and a lid domain that encloses the substrates. The lid domain is thought to close over the TIM-barrel domain forming contacts with the substrates to promote catalysis and may be involved in stabilising the activated state when the allosteric activator is bound. However, it remains unknown whether the lid domain is essential for pyruvate kinase catalytic or regulatory function. To address this, we removed the lid domain of Escherichia coli pyruvate kinase type 1 (PK TIM+Reg ) using protein engineering. Biochemical analyses demonstrate that, despite the absence of key catalytic residues in the lid domain, PK TIM+Reg retains a low level of catalytic activity and has a reduced binding affinity for the substrate phosphoenolpyruvate. The enzyme retains allosteric activation, but the regulatory profile of the enzyme is changed relative to the wild-type enzyme. Analytical ultracentrifugation and small-angle X-ray scattering data show that, beyond the loss of the lid domain, the PK TIM+Reg structure is not significantly altered and is consistent with the wild-type tetramer that is assembled through interactions at the TIM and regulatory domains. Our results highlight the contribution of the lid domain for facilitating pyruvate kinase catalysis and regulation, which could aid in the development of small molecule inhibitors for pyruvate kinase and related lid-regulated enzymes.

Human pathogenic and commensal bacteria have evolved the ability to scavenge host-derived sialic acids and subsequently degrade them as a source of nutrition. Expression of the Escherichia coli yjhBC operon is controlled by the repressor protein nanR, which regulates the core machinery responsible for the import and catabolic processing of sialic acid. The role of the yjhBC encoded proteins is not known-here, we demonstrate that the enzyme YjhC is an oxidoreductase/dehydrogenase involved in bacterial sialic acid degradation. First, we demonstrate in vivo using knockout experiments that YjhC is broadly involved in carbohydrate metabolism, including that of N-acetyl-d-glucosamine, N-acetyl-d-galactosamine and N-acetylneuraminic acid. Differential scanning fluorimetry demonstrates that YjhC binds N-acetylneuraminic acid and its lactone variant, along with NAD(H), which is consistent with its role as an oxidoreductase. Next, we solved the crystal structure of YjhC in complex with the NAD(H) cofactor to 1.35 Å resolution. The protein fold belongs to the Gfo/Idh/MocA protein family. The dimeric assembly observed in the crystal form is confirmed through solution studies. Ensemble refinement reveals a flexible loop region that may play a key role during catalysis, providing essential contacts to stabilize the substrate-a unique feature to YjhC among closely related structures. Guided by the structure, in silico docking experiments support the binding of sialic acid and several common derivatives in the binding pocket, which has an overall positive charge distribution. Taken together, our results verify the role of YjhC as a bona fide oxidoreductase/dehydrogenase and provide the first evidence to support its involvement in sialic acid metabolism., (© 2019 Wiley Periodicals, Inc.)

The fitness effects of mutations can depend on the genetic backgrounds in which they occur and thereby influence future opportunities for evolving populations. In particular, mutations that fix in a population might change the selective benefit of subsequent mutations, giving rise to historical contingency. We examine these effects by focusing on mutations in a key metabolic gene, pykF, that arose independently early in the history of 12 Escherichia coli populations during a long-term evolution experiment. Eight different evolved nonsynonymous mutations conferred similar fitness benefits of ∼10% when transferred into the ancestor, and these benefits were greater than the one conferred by a deletion mutation. In contrast, the same mutations had highly variable fitness effects, ranging from ∼0% to 25%, in evolved clones isolated from the populations at 20,000 generations. Two mutations that were moved into these evolved clones conferred similar fitness effects in a given clone, but different effects between the clones, indicating epistatic interactions between the evolved pykF alleles and the other mutations that had accumulated in each evolved clone. We also measured the fitness effects of six evolved pykF alleles in the same populations in which they had fixed, but at seven time points between 0 and 50,000 generations. Variation in fitness effects was high at intermediate time points, and declined to a low level at 50,000 generations, when the mean fitness effect was lowest. Our results demonstrate the importance of genetic context in determining the fitness effects of different beneficial mutations even within the same gene., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.)

Dihydrodipicolinate synthase (DHDPS) catalyzes the first committed step of the lysine biosynthetic pathway. The tetrameric structure of DHDPS is thought to be essential for enzymatic activity, as isolated dimeric mutants of Escherichia coli DHDPS possess less than 2.5% that of the activity of the wild-type tetramer. It has recently been proposed that the dimeric form lacks activity due to increased dynamics. Tetramerization, by buttressing two dimers together, reduces dynamics in the dimeric unit and explains why all active bacterial DHDPS enzymes to date have been shown to be homo-tetrameric. However, in this study we demonstrate for the first time that DHDPS from methicillin-resistant Staphylococcus aureus (MRSA) exists in a monomer-dimer equilibrium in solution. Fluorescence-detected analytical ultracentrifugation was employed to show that the dimerization dissociation constant of MRSA-DHDPS is 33 nm in the absence of substrates and 29 nm in the presence of (S)-aspartate semialdehyde (ASA), but is 20-fold tighter in the presence of the substrate pyruvate (1.6 nm). The MRSA-DHDPS dimer exhibits a ping-pong kinetic mechanism (k(cat)=70+/-2 s(-1), K(m)(Pyruvate)=0.11+/-0.01 mm, and K(m)(ASA)=0.22+/-0.02 mm) and shows ASA substrate inhibition with a K(si)(ASA) of 2.7+/-0.9 mm. We also demonstrate that unlike the E. coli tetramer, the MRSA-DHDPS dimer is insensitive to lysine inhibition. The near atomic resolution (1.45 A) crystal structure confirms the dimeric quaternary structure and reveals that the dimerization interface of the MRSA enzyme is more extensive in buried surface area and noncovalent contacts than the equivalent interface in tetrameric DHDPS enzymes from other bacterial species. These data provide a detailed mechanistic insight into DHDPS catalysis and the evolution of quaternary structure of this important bacterial enzyme.