This study showed the crystal structures of MccB from
S.
aureus in the presence and absence of PLP at the active site. The overall structures of the bacterial MccB shared structural organization with human CGL and its homologues, MetB and MetC, in bacteria (
Supplementary Materials Figure S3). A structural comparison of the apo- and PLP-bound structures of SaMccB showed that the inter-subunit loop and the α-helical flap region have substantially different conformational constraints. The PLP-bound structures became flexible in the α-helical flap region; this was different from MetC, which showed a stable interaction regardless of PLP binding (
Figure 4b,d).
Delocalized electrons by conjugated double bonds and the electron sink at the N1-atom of PLP are essential for most PLP-mediated reactions. The Lys residue for PLP binding is commonly used as the first and primary general acid/base when it is free from PLP binding. However, the other general acid/base at the active site residues might also be important in determining which reactions are preferred in the individual enzyme. By comparing the reaction processes between the γ-elimination of MccB and the β-elimination of MetB (
Figure 5), we identified the importance of the second base residue (Base in
Figure 5b) for deprotonation at the β-carbon in MccB, which is a prerequisite for γ-elimination. The resulting carbanion at Cβ was stabilized by N
SB through a new pi-bond between Cβ and Cγ. We noted a wider active site of SaMccB, which might prefer deprotonation at Cβ. Because the N
SB atom of PLP turned from sp2 to sp3 orbital, and the pseudo-planar conjugated double bond system to the pyridine ring was broken at N
SB, more room was required to hold this intermediate. The structural flexibility of MccB could better compensate for this conformational change during the γ-elimination reaction.
What would be the second base residue in MccB? We noted the Ser–water–Ser–Glu hydrogen bond network at the active site of MccB. According to the model structure of the external aldimine (step II in
Figure 5b and
Figure 3c), Oγ of Ser323 was near the Cβ atom of the Schiff base nitrogen atom of PLP, within 3.5 Å. Glu328 was surrounded in a hydrophobic environment and was connected to Ser323 via Ser202 and the water molecule (
Figure 3c). Because the pKa value of Glu328 increased due to its hydrophobic environment, the proton of Ser328 was easily transferred in this network. This hydrogen bond network was comparable with the catalytic triad, Ser–His–Asp, in traditional serine proteases, where the proton of Ser is transferred to Asp under a hydrophobic environment via His.
S. aureus Mu50 (ATCC 700699) is a vancomycin intermediate
S. aureus that displays robust virulence properties. It causes severe infectious diseases ranging from mild infections, such as skin infections and food poisoning, to life-threatening infections, such as sepsis, endocarditis, and toxic shock syndrome [
19]. However, because of bacterial resistance, the prognosis for
S. aureus infection is still poor despite early diagnosis and appropriate treatment [
20]. Although there are various theories regarding how
S. aureus acquires its antibiotic resistance, very little is known. In this regard, understanding the bacterial defense mechanisms of
S. aureus against antibiotics is crucial. Many bactericidal antibiotics kill the bacteria by stimulating the production of highly toxic hydroxyl radicals, whose production is mediated by Fe
2+ [
21]. To cope with this stress, the bacteria could alter gene expression levels and metabolism, which are possibly linked to staphylococcal virulence factor synthesis and inhibition of the production of hydroxyl radicals [
11]. SaMccB (named YhrB in the reference) is commonly listed as a gene that is downregulated by ampicillin, kanamycin, and norfloxacin. Because free cysteine could accelerate the Fe
2+-mediated Fenton reaction by reducing Fe
3+ to Fe
2+ [
22], the downregulation of SaMccB in response to diverse antibiotics could be explained. To control the resistance of
S. aureus, a better understanding of metabolic enzymes is important. Our study provided molecular clues into how homologous enzymes using PLP as the cofactor catalyze different reactions of metabolic enzymes, whose gene expression is regulated by antibiotic stress. Further research will promote a better understanding of how bacteria can defend against conditions of stress.