Even though there are many historical studies of the enzymes associated with the biosynthesis of CoA, the cloning and characterisation of all the enzymes in the pathway are reasonably recent events. In 1979, the gene encoding PanK in Salmonella typhimuriun was the first encoding gene of these pathway enzymes to be identified [100]. Subsequently, in 1992, PanK from E. coli was the first enzyme of CoA biosynthesis to be cloned and expressed [101]. The last enzyme of the pathway to be cloned was PPCS from E. coli, which occurred in 2001 [102]. However, thenceforth there has been immense development in the comprehension of the enzymes in this biosynthetic pathway.
2.2.3.1 Pantothenate kinase
PanK, the gene product of coaA or coaX, is the best studied of all the enzymes involved in the biosynthesis of CoA. After the first PanK (that of E. coli) was expressed and purified, the PanKs of several other organisms that represented mammal, plant, fungal, and eubacterial sources, followed [66]. It has recently been identified that PanKs, regardless of their catalytic function, can be subdivided into three main types, namely PanK type I, type II and type III. This differentiation is based on structural fold, sequence identity, as well as inhibition and
20 catalytic characteristics [103]. It was first assumed that the differentiation between the PanKs was along the phylogenetic lines, which noticeably defined prokaryotic and eukaryotic types. This notion of simple classification has since been discarded, as it has become evident that certain bacterial PanKs are closer related to previously assumed eukaryotic enzymes than to other bacteria [103]. In addition, some organisms possess two different PanK types, such as Mycobacterium tuberculosis [104].
PanK type I - The type I PanK of E. coli is the best representative of these enzymes as it was the first to be fully characterised and was regarded as the ‗prototypical‘ bacterial PanK [101]. This enzyme is encoded by the coaA gene and exists as a homodimer [105, 106]. Furthermore, its structure suggests that the type I PanKs belong to the P-loop kinase family, which is a defining characteristic of this type of PanK [107, 108]. In addition to the highly cooperative binding of ATP, the binding of substrates also follow an ordered sequential mechanism, where ATP binds prior to pantothenate [106].
Another significant characteristic of type I PanKs is their particularly flexible substrate- binding sites. The ATP-binding site has been shown to allow competitive inhibition with the binding of CoA (and CoA thioesters, albeit less potent) [109, 110]. This facilitates feedback inhibition with the final product of the pathway, which is important for regulating intracellular CoA concentrations. Similarly, the low-level substrate specificity exhibited by the pantothenate-binding site enables PanK type I to phosphorylate various types of pantothenate analogues including PantSH [111].
PanK type II - The type II PanKs are mostly harboured by eukaryotic organisms, such as plants [112, 113], fungi [114], mammals [115, 116], and insects [117]. Several of these organisms possess numerous PanK-encoding genes, where each gene is expressed in a specific tissue. In humans, for example, there are four main encoding genes, namely PANK1, PANK2, PANK3, and PANK4 [66]. PANK1 is mainly expressed in the kidney and liver and less in the muscle and heart, while PANK2 is expressed in most tissues and localises in the mitochondria [118, 119]. Conversely, PANK3 is highly expressed in the liver and to a lesser extent in the skeletal muscle and heart, whereas PANK4 expression is located in the heart and muscle [120]. Moreover, some of these genes can encode two related but different PanK isoforms. Examples of this include PANK1 of humans and mice, which has PanK1α and PanK1β as protein products [121, 122].
Type II PanKs do share some similarities with type I PanKs with regards to substrate specificity and inhibition. The type II PanKs can also undergo feedback inhibition by CoA biosynthesis end products. However, in type II PanKs, the CoA thioesters (acetyl-CoA in
21 particular) are usually more potent inhibitors, compared to free CoA [116, 122–124]. Similar to type I PanKs, these PanK types also accept pantothenamides (amide containing analogues of pantothenate) as substrate alternatives [125]. Nevertheless, type I and type II PanKs differ in their primary sequence profiles and structural folds. The type II PanKs are not members of the P-loop kinase family like their type I counterparts. In fact, the structures of various type II PanKs suggest that these enzymes belong to the sugar kinase/heat-shock protein 70/actin (ASKHA) superfamily of kinases [107, 126, 127].
Additionally, it has been demonstrated that some Gram-positive bacteria possess PanK-encoding genes that are related more closely to the ‗eukaryotic‘ type II PanKs (described above) than to the ‗prototypical‘ bacterial type I PanKs, based on primary sequence homology. The enzyme of Staphylococcus aureus is one of these bacterial type II PanKs [128, 129]. This enzyme, however, is not affected by feedback inhibition by CoA and its thioesters like its eukaryotic counterparts [128, 129]. Yet, in spite of this, the PanK of S. aureus still accepts pantothenamides as substrate alternatives [125, 128].
PanK type III - The suggestion that a third PanK type exists first arose following genome- wide analyses where gene homologues encoding all the CoA biosynthetic enzymes, except PanK, were identified in some bacteria, such as Pseudomonas aeruginosa and Helicobacter pylori [83, 130]. The results remained the same irrespective of submitting type I PanK or any type II PanK in the homology searches. Following this, a PanK-encoding gene, distinguishable from other PanK types, was reported in Bacillus subtillus [131]. This gene was termed coaX in order to differentiate between it and the more popular coaA gene. Subsequently, homology searches based on the coaX sequence identified homologues in other bacteria that were initially thought to have apparent missing PanK enzymes [66]. Interestingly, this gene is exclusively present in a broad range of pathogenic bacteria [132]. After the expression, purification and full characterisation of the CoaX protein of B. subtillus and H. pylori, it was revealed that these PanKs demonstrated a noticeable difference in kinetic parameters compared to that of the other PanK types, in addition to the variation in primary sequence and structure [133]. For instance, even though the affinity for pantothenate of these enzymes were relatively the same as that of the other PanKs, their Km
values for ATP were reported to be in the millimolar range (up to ~10 mM for H. pylori) compared to the micromolar ranges for type I & II PanKs. Furthermore, it was also shown that these PanKs do not experience feedback inhibition by CoA or acetyl-CoA nor do they accept pantothenamides as alternative substrates [103, 127, 133]. Thus, PantSH can also not be accepted as a substrate, which implies that the salvage pathway (Red box, Scheme 2.2) is not an option in the organisms containing a type III PanK. On the basis of
22 these attributes, the CoaX proteins were recognised as the third type of PanK, and thus named type III PanKs. Moreover, structural studies have established that the type III PanKs are also members of the ASKHA superfamily like their type II counterparts [103, 127, 132].
2.2.3.2 Phosphopantothenoylcysteine synthetase
Two main forms of PPCS have been identified to date, specifically bacterial PPCS and eukaryotic PPCS. The bacterial PPCS utilises CTP to activate the carboxylate of 4‘-phosphopantothenate (2.8, Scheme 2.2) by forming an acyl-cytidylate intermediate [102]. This bacterial version of PPCS typically forms a fusion protein with the subsequent enzyme in the pathway, PPCDC, which produces the bifunctional CoaBC protein (protein product of coaBC). Conversely, the eukaryotic form of the enzyme (protein product of coaB) is monofunctional and utilises ATP to activate the substrate carboxylate [134–136]. This also seems to be the case in Streptococci and Enterococci, as they are predicted to be exceptions to the bacterial bifunctional enzyme formation [130]. However, both the eukaryotic and bacterial PPCS exhibit dimeric structures with similar folds [137].
Following the expression, purification and characterisation of the bacterial CoaBC protein, it was reported to have a Km value of 55 µM for 4‘-phosphopantothenate, with a Km value of
106 µM and 109 µM for CTP and L-cysteine, respectively [102, 138]. Studies have also shown that the product of PPCS in the CoaBC fusion protein, phosphopantothenoylcysteine (2.9, Scheme 2.2), dissociates from the protein and then binds to a separate active site on the PPCDC domain to commence further transformation [139]. This suggests that the merging of these two enzymes in bacteria offer no extensive functional significance. This fusion does, however, ensure that the concentration of the PPCS and PPCDC enzymes remain equal [139].
PPCS has also been shown to have a very high selectivity for cysteine [140]. In a study by Strauss et al. [140], the enzyme was exposed to 500 000-fold excess serine, which has a structure that closely resembles that of cysteine, but only cysteine was integrated into the product. This selectivity for cysteine is not surprising, as the structure of serine has a thiol group instead of the hydroxyl group from cysteine. Therefore, if the condensation of serine proceeds, it could result in the formation of oxy-CoA, a potential toxic CoA analogue [141].
2.2.3.3 Phosphopantothenoylcysteine decarboxylase
As mentioned previously, prokaryotic PPCS and PPCDC enzymes fuse to form a bifunctional CoaBC protein product, with the exceptions of Streptococci and Enterococci, which supposedly possess monofunctional enzymes. These prokaryotic CoaBC enzymes share a structural feature with the lantibiotic decarboxylases, which is that they form
23 homododecamers (tetramers of trimers) [142, 143]. This occurs via interactions of the CoaC domains. However, the eukaryotic PPCDC enzymes are reported to be monofunctional proteins with a trimeric structure [144].
The activity of the PPCDC enzyme has proved to be far more elusive than all the other enzymes in the CoA pathway, concerning both its mechanism and identification. The enzymatic mechanism was finally solved in 2000, after the characterisation of the PPCDC domain associated with the E. coli CoaBC protein [143]. The reaction catalysed by PPCDC causes the formation of a negative charge on the carbon next to the amide of phosphopantothenoylcysteine. This charge is then stabilised by a flavin mononucleotide cofactor (tightly bound to the enzyme), which is responsible for the oxidation of the substrate cysteine thiol. Subsequently, a thioaldehyde is formed, followed by its spontaneous decarboxylation to produce an enethiol, which is then reduced back to yield PPantSH (2.10, Scheme 2.2). This last step relies on an active site cysteine, which was reported to be absent in the CoaBC protein of Methanocaldococcus jannaschii and instead, it was replaced by a glutamic acid [145]. Consequently, this indicated that PPCDC enzymes may exhibit variations in their mechanisms.
2.2.3.4 Phosphopantetheine adenylyltransferase
PPAT, also known as the CoaD protein (protein product of coaD), is the enzyme responsible for catalysing the penultimate step in the CoA biosynthetic pathway. In bacteria, PPAT is reported to be a single enzyme [146]. This enzyme adopts a homohexameric structure, which consists of a dimer of separate trimers [147–150]. Following a comprehensive kinetic characterisation of the E. coli PPAT, it was revealed that this enzyme follows an arbitrary bi-bi mechanism in which a ternary complex of ATP, PPantSH and enzyme is formed [151]. The Km values that were determined are 4.7 ± 0.5 µM for PPantSH and 220 ± 10 µM for
ATP. In addition, it was also reported that the bacterial PPAT experiences feedback inhibition. DePCoA (2.11, Scheme 2.2) binding was revealed to be competitive with both PPantSH and ATP binding; whereas the binding of pyrophosphate was competitive with ATP (pyrophosphate product inhibition of PPantSH binding could not be determined). The binding of CoA is also competitive with PPantSH, ATP and DePCoA. Furthermore, CoA thioesters are much weaker inhibitors compared to free CoA, suggesting that CoA is the main inhibitor in feedback inhibition of this enzyme. According to structural studies of bacterial PPAT, they exhibit sequence homology to members of the nucleotidyltransferase α/β phosphodiesterase enzyme superfamily [148].
In contrast, the PPAT activity in most eukaryotes is fused to DPCK, which is the last enzyme in the pathway [152]. This produces a bifunctional PPAT/DPCK protein, better known as
24 CoA synthase (CoASy). The sequences of these eukaryotic bifunctional proteins have been shown to have little similarity with that of the monofunctional bacterial enzymes [134, 135, 153, 154]. There is also little known about the structure of the PPAT domain in the CoASy protein. However, preliminary studies propose that they too belong to the superfamily of nucleotidyltransferase α/β phosphodiesterases [155].
2.2.3.5 Dephospho-coenzyme A kinase
As mentioned previously, the DPCK activity in eukaryotes is fused to PPAT as part of the CoASy bifunctional protein [152]. However, in contrast to PPAT, the sequence of the DPCK domain of CoASy demonstrates good homology with its prokaryotic counterpart, which is reported to be a monofunctional protein [135, 153, 154]. The expression and characterisation of the E. coli DPCK protein (protein product of coaE) revealed that it is a 22.6 kDa monomer in solution with Km values reported being 740 µM and 140 µM for
DePCoA and ATP, respectively [156]. Conversely, the DPCK domain of the CoASy protein demonstrated a similar affinity for ATP (reported Km value of 192 µM) but a much higher
affinity for DePCoA (reported Km value of 5.2 ± 1.5 µM) [154]. This results in the irreversible
PPAT activity of the bifunctional protein, which is normally reversible as a monofunctional protein. The structural analyses of numerous DPCK enzymes, including the monofunctional protein of E. coli and the CoASy DPCK domain of Mus musculus, revealed that they belong to the P-loop kinase enzyme family [107, 157].
There are still some uncertainties regarding the kinetic parameters of DPCK, due to the limited information available on this enzyme. Although very little is known about the catalytic mechanism of DPCK, it has been proposed that a conformational change may take place during catalyses. The crystal structures of E. coli DPCK (bound to ADP only) and Thermotoga maritima DPCK (bound to DePCoA and ADP) were superimposed to reveal a distinct difference in conformation between the two enzymes [157]. This change suggested that the enzyme adopts a closed conformation after binding to DePCoA in order to bring it closer to ATP catalytic activity. In fact, similar substrate-induced conformational changes are not uncommon in the P-loop kinase enzyme family [158].
If this conformational change-hypothesis is true for E. coli DPCK, it may explain the low substrate affinity for DePCoA observed in the in vitro studies, as the initial binding of the substrate has a low affinity, followed by the conformational change to a structure with a higher affinity to allow catalysis to proceed. Alternatively, since the DPCK of E. coli has been shown to be a monomer [156] and yet in the presence of sulphate it crystallises as a trimer [157]; the apparent low affinity observed for DePCoA could be due to the changes in quaternary protein structure.
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