Functional and structural properties of pyridoxal reductase (PdxI) from Escherichia coli: a pivotal enzyme in the vitamin B6 salvage pathway.

Pyridoxine 4-dehydrogenase (PdxI), a NADPH-dependent pyridoxal reductase, is one of the key players in the Escherichia coli pyridoxal 5'-phosphate (PLP) salvage pathway. This enzyme, which catalyses the reduction of pyridoxal into pyridoxine, causes pyridoxal to be converted into PLP via the formation of pyridoxine and pyridoxine phosphate. The structural and functional properties of PdxI were hitherto unknown, preventing a rational explanation of how and why this longer, detoured pathway occurs, given that, in E. coli, two pyridoxal kinases (PdxK and PdxY) exist that could convert pyridoxal directly into PLP. Here, we report a detailed characterisation of E. coli PdxI that explains this behaviour. The enzyme efficiently catalyses the reversible transformation of pyridoxal into pyridoxine, although the reduction direction is thermodynamically strongly favoured, following a compulsory-order ternary-complex mechanism. In vitro, the enzyme is also able to catalyse PLP reduction and use NADH as an electron donor, although with lower efficiency. As with all members of the aldo-keto reductase (AKR) superfamily, the enzyme has a TIM barrel fold; however, it shows some specific features, the most important of which is the presence of an Arg residue that replaces the catalytic tetrad His residue that is present in all AKRs and appears to be involved in substrate specificity. The above results, in conjunction with kinetic and static measurements of vitamins B6 in cell extracts of E. coli wild-type and knockout strains, shed light on the role of PdxI and both kinases in determining the pathway followed by pyridoxal in its conversion to PLP, which has a precise regulatory function.

X-ray crystallography and sickle cell disease drug discovery-a tribute to Donald Abraham.

X-ray crystallography and structure-based drug discovery have played a major role in the discovery of antisickling agents that target hemoglobin (Hb) for the treatment of sickle cell disease (SCD). Sickle cell disease, the most common inherited hematologic disorder, occurs as a result of a single point mutation of ßGlu6 in normal human adult hemoglobin (HbA) to ßVal6 in sickle hemoglobin (HbS). The disease is characterized by polymerization of HbS and sickling of red blood cells (RBCs), leading to several secondary pathophysiologies, including but not limited to vaso-occlusion, hemolytic anemia, oxidative stress, inflammation, stroke, pain crisis, and organ damage. Despite the fact that SCD was the first disease to have its molecular basis established, the development of therapies was for a very long time a challenge and took several decades to find therapeutic agents. The determination of the crystal structure of Hb by Max Perutz in the early 60s, and the pioneering X-ray crystallography research by Donald J. Abraham in the early 80s, which resulted in the first structures of Hb in complex with small molecule allosteric effectors of Hb, gave much hope that structure-based drug discovery (SBDD) could be used to accelerate development of antisickling drugs that target the primary pathophysiology of hypoxia-induced HbS polymerization to treat SCD. This article, which is dedicated to Donald J. Abraham, briefly reviews structural biology, X-ray crystallography and structure-based drug discovery from the perspective of Hb. The review also presents the impact of X-ray crystallography in SCD drug development using Hb as a target, emphasizing the major and important contributions by Don Abraham in this field.

Characterization of the Escherichia coli pyridoxal 5'-phosphate homeostasis protein (YggS): Role of lysine residues in PLP binding and protein stability.

The pyridoxal 5'-phosphate (PLP) homeostasis protein (PLPHP) is a ubiquitous member of the COG0325 family with apparently no catalytic activity. Although the actual cellular role of this protein is unknown, it has been observed that mutations of the PLPHP encoding gene affect the activity of PLP-dependent enzymes, B6 vitamers and amino acid levels. Here we report a detailed characterization of the Escherichia coli ortholog of PLPHP (YggS) with respect to its PLP binding and transfer properties, stability, and structure. YggS binds PLP very tightly and is able to slowly transfer it to a model PLP-dependent enzyme, serine hydroxymethyltransferase. PLP binding to YggS elicits a conformational/flexibility change in the protein structure that is detectable in solution but not in crystals. We serendipitously discovered that the K36A variant of YggS, affecting the lysine residue that binds PLP at the active site, is able to bind PLP covalently. This observation led us to recognize that a number of lysine residues, located at the entrance of the active site, can replace Lys36 in its PLP binding role. These lysines form a cluster of charged residues that affect protein stability and conformation, playing an important role in PLP binding and possibly in YggS function.

Design, Synthesis, and Investigation of Novel Nitric Oxide (NO)-Releasing Aromatic Aldehydes as Drug Candidates for the Treatment of Sickle Cell Disease.

Sickle cell disease (SCD) is caused by a single-point mutation, and the ensuing deoxygenation-induced polymerization of sickle hemoglobin (HbS), and reduction in bioavailability of vascular nitric oxide (NO), contribute to the pathogenesis of the disease. In a proof-of-concept study, we successfully incorporated nitrate ester groups onto two previously studied potent antisickling aromatic aldehydes, TD7 and VZHE039, to form TD7-NO and VZHE039-NO hybrids, respectively. These compounds are stable in buffer but demonstrated the expected release of NO in whole blood in vitro and in mice. The more promising VZHE039-NO retained the functional and antisickling activities of the parent VZHE039 molecule. Moreover, VZHE039-NO, unlike VZHE039, significantly attenuated RBC adhesion to laminin, suggesting this compound has potential in vivo RBC anti-adhesion properties relevant to vaso-occlusive events. Crystallographic studies show that, as with VZHE039, VZHE039-NO also binds to liganded Hb to make similar protein interactions. The knowledge gained during these investigations provides a unique opportunity to generate a superior candidate drug in SCD with enhanced benefits.

Mechanistic Analysis of the VirA Sensor Kinase in Agrobacterium tumefaciens Using Structural Models.

Agrobacterium tumefaciens pathogenesis of plants is initiated with signal reception and culminates with transforming the genomic DNA of its host. The histidine sensor kinase VirA receives and reacts to discrete signaling molecules for the full induction of the genes necessary for this process. Though many of the components of this process have been identified, the precise mechanism of how VirA coordinates the response to host signals, namely phenols and sugars, is unknown. Recent advances of molecular modeling have allowed us to test structure/function predictions and contextualize previous experiments with VirA. In particular, the deep mind software AlphaFold has generated a structural model for the entire protein, allowing us to construct a model that addresses the mechanism of VirA signal reception. Here, we deepen our analysis of the region of VirA that is critical for phenol reception, model and probe potential phenol-binding sites of VirA, and refine its mechanism to strengthen our understanding of A. tumefaciens signal perception.

Design, Synthesis, and Antisickling Investigation of a Nitric Oxide-Releasing Prodrug of 5HMF for the Treatment of Sickle Cell Disease.

5-hydroxyfurfural (5HMF), an allosteric effector of hemoglobin (Hb) with an ability to increase Hb affinity for oxygen has been studied extensively for its antisickling effect in vitro and in vivo, and in humans for the treatment of sickle cell disease (SCD). One of the downstream pathophysiologies of SCD is nitric oxide (NO) deficiency, therefore increasing NO (bio)availability is known to mitigate the severity of SCD symptoms. We report the synthesis of an NO-releasing prodrug of 5HMF (5HMF-NO), which in vivo, is expected to be bio-transformed into 5HMF and NO, with concomitant therapeutic activities. In vitro studies showed that when incubated with whole blood, 5HMF-NO releases NO, as anticipated. When incubated with sickle blood, 5HMF-NO formed Schiff base adduct with Hb, increased Hb affinity for oxygen, and prevented hypoxia-induced erythrocyte sickling, which at 1 mM concentration were 16%, 10% and 27%, respectively, compared to 21%, 18% and 21% for 5HMF. Crystal structures of 5HMF-NO with Hb showed 5HMF-NO bound to unliganded (deoxygenated) Hb, while the hydrolyzed product, 5HMF bound to liganded (carbonmonoxy-ligated) Hb. Our findings from this proof-of-concept study suggest that the incorporation of NO donor group to 5HMF and analogous molecules could be a novel beneficial strategy to treat SCD and warrants further detailed in vivo studies.