A Subfamily of Bacterial Ribokinases Utilizes a Hemithioacetal for Pyridoxal Phosphate Salvage

Abstract

Pyridoxal 5'-phosphate (PLP) is the active vitamer of vitamin B6 and acts as an essential cofactor in many aspects of amino acid and sugar metabolism. The virulence and survival of pathogenic bacteria such as Mycobacterium tuberculosis depend on PLP, and deficiencies in humans have also been associated with neurological disorders and inflammation. While PLP can be synthesized by a de novo pathway in bacteria and plants, most higher organisms rely on a salvage pathway that phosphorylates either pyridoxal (PL) or its related vitamers, pyridoxine (PN) and pyridoxamine (PM). PL kinases (PLKs) are essential for this phosphorylation step and are thus of major importance for cellular viability. We recently identified a pyridoxal kinase (SaPLK) as a target of the natural product antibiotic rugulactone (Ru) in Staphylococcus aureus. Surprisingly, Ru selectively modified SaPLK not at the active site cysteine, but on a remote cysteine residue. Based on structural and biochemical studies, we now provide insight into an unprecedented dual Cys charge relay network that is mandatory for PL phosphorylation. The key component is the reactive Cys 110 residue in the lid region that forms a hemithioactetal intermediate with the 4'-aldehyde of PL. This hemithioacetal, in concert with the catalytic Cys 214, increases the nucleophilicity of the PL 5'-OH group for the inline displacement reaction with the γ-phosphate of ATP. A closer inspection of related enzymes reveals that Cys 110 is conserved and thus serves as a characteristic mechanistic feature for a dual-function ribokinase subfamily herein termed CC-PLKs.

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“…The ferredoxin protein has been reported in many plants and plastid enzymes are derived from electronic ferredoxin, including sulfur reductase, fatty acid desaturase, nitrogen reductase and thioredoxin reductase46. Pyridoxine phosphate oxidase is involved in the de novo synthesis of vitamin B6 in plants in one oxidase pathway, which catalyzes the phosphorylation of pyridoxine (PNP) and then pyridoxamine phosphate (PMP) is oxidized to pyridoxal phosphate (PLP)47. Molecular chaperones are members of a large family of proteins in cells48.…”

Essential Metabolic Routes as a Way to ESKAPE From Antibiotic Resistance

“…In terms of structural biology, some crystal structures of ThiD are available by the time of writing of this paper, including the enzymes from Interestingly, the S. aureus pyridoxal kinase enzyme (SaPdxK) has a dual role, phosphorylating pyridoxal and pyridoxine in the pyridoxal de novo biosynthesis pathway as well as HMP in the thiamin biosynthesis pathway, with a K M almost 20 times greater for HMP than for pyridoxal and k cat values three times faster for pyridoxal (24). So, SaPdxK has some redundancy with S. aureus ThiD (SaThiD), with less efficiency, though.…”

Essential Metabolic Routes as a Way to ESKAPE from Antibiotic Resistance

“…Interestingly, the S. aureus ThiD enzyme (SaThiD) has a dual role, phosphorylating HMP in the thiamin biosynthesis pathway as well as pyridoxal and pyridoxine in the pyridoxal biosynthesis pathway, with a K M almost 20 times greater for HMP than for pyridoxal and k cat values 3 times faster for pyridoxal (21), suggesting that SaThiD is more efficient as a pyridoxal kinase than as an HMP kinase. SaThiD was shown to be inhibited by Rugulactone, a natural product and, in the absence of thiamine in the medium, the MIC observed for Rugulactone was four times lower than in the presence of thiamine (22), suggesting that the inhibitory effect of Rugulactone is due to ThiD inhibition, although Rugulactone also inhibits other kinases.…”

“…Similar to what is observed to the thiamine biosynthesis pathway, humans lack the genes for Pdx1 and Pdx2, making the development of specific antibiotic candidates an attracting strategy. In the same direction, for some pathogenic organisms such as Helicobacter pylori (32), Mycobacterium tuberculosis (33), and Streptococcus pneumoniae (34), the depletion in vitamin B6 resulted in reduced virulence (21), indicating that the biosynthesis of this vitamin may be a good strategy for the design of new antibiotic candidates.…”

“…These kinases act phosphorylating the vitamers pyridoxal, pyridoxine and pyridoxamine into their phosphorylated forms (20,25,42). Interestingly, in some organisms, including pathogenic microorganisms such as S. aureus, the PdxK activity is carried out by the HMP kinase ThiD, showing some functional convergence between the vitamin B6 and B1 pathways (21). Not surprisingly, this enzyme has also been shown to be validated druggable target for some pathogens, such as Trypanosoma brucei for example (43), although specificity may be an important issue, since humans also have genes for PdxK known to be inhibited by drugs like theophylline, for example, with known neurotoxic effects (44).…”