Chemical Proteomics Reveal the Inventory of Pyrroloquinoline Quinone Binding Proteins in Bacteria

Pyrroloquinoline quinone (PQQ) is a bacterial
redox
cofactor enabling enzyme catalysis in various sugar and alcohol dehydrogenases.
However, its proposed additional role as a “longevity vitamin”
lacks a clear molecular basis and is thus highly debated. Here, we
applied chemical proteomics to identify previously unknown classes
of PQQ-binding proteins. We designed and synthesized
a structurally diverse suite of five PQQ probes equipped
with a diazirine photo-cross-linker and an alkyne handle for target
identification. The fidelity of the probes was first evaluated for
two well-characterized bacterial PQQ-dependent enzymes,
demonstrating not only probe binding but also the reconstitution of
catalytic activity. We then commenced with proteome profiling of Escherichia coli and Pseudomonas putida cells and unraveled a distinct set of putative PQQ-binding
proteins. Recombinant expression of selected hits, including several
chaperones, validated PQQ binding. Notably, in some cases, PQQ even formed covalent adducts with selected lysine residues,
for instance, in the AAA+ ATPase RuvB involved in DNA remodeling.
Overall, our work highlights the utility of PQQ probes
to further unravel the complement of cofactor-binding proteins in
whole cells. It also provides a basis for future mechanistic studies
of PQQ functions beyond redox catalysis.

吡咯并喹啉醌（Pyrroloquinoline quinone, PQQ）是一类细菌氧化还原辅因子，可介导多种糖脱氢酶与醇脱氢酶的酶催化反应。
然而，此前提出的其作为“长寿维生素”的额外功能，尚无明确的分子机制支撑，因此争议颇大。
本研究中，我们运用化学蛋白质组学方法，鉴定出此前未知的PQQ结合蛋白类别。
我们设计并合成了一组结构多样的PQQ探针，共五种，均带有重氮光交联基团与炔基标签，用于靶标蛋白的鉴定。
首先针对两种已被充分表征的细菌PQQ依赖型酶，对探针的保真度进行了评估，结果显示探针不仅可实现特异性结合，还能重建其催化活性。
随后我们对大肠杆菌（Escherichia coli）与恶臭假单胞菌（Pseudomonas putida）的细胞进行蛋白质组分析，揭示了一组独特的推定PQQ结合蛋白。
对筛选得到的候选蛋白（包括多种分子伴侣）进行重组表达后，验证了其与PQQ的结合能力。
值得注意的是，在部分案例中，PQQ甚至可与特定赖氨酸残基形成共价加合物，例如在参与DNA重塑的AAA+ ATP酶RuvB中。
综上，本研究证明了PQQ探针可用于进一步解析全细胞内的辅因子结合蛋白组，同时也为后续研究PQQ在氧化还原催化之外的功能机制提供了坚实基础。