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Acetone carboxylases (ACs) catalyze the metal- and ATP-dependent conversion of acetone and bicarbonate to form acetoacetate. Interestingly, two homologous ACs that have been biochemically characterized have been reported to have different metal complements, implicating different metal dependencies in catalysis. ACs from proteobacteria Xanthobacter autotrophicus and Aromatoleum aromaticum share 68% sequence identity but have been proposed to have different catalytic metals. In this work, the two ACs were expressed under the same conditions in Escherichia coli and were subjected to parallel chelation and reconstitution experiments with Mn(II) or Fe(II). Electron paramagnetic and Mössbauer spectroscopies identified signatures, respectively, of Mn(II) or Fe(II) bound at the active site. These experiments showed that the respective ACs, without the assistance of chaperones, second metal sites, or post-translational modifications facilitate correct metal incorporation, and despite the expected thermodynamic preference for Fe(II), each preferred a distinct metal. Catalysis was likewise associated uniquely with the cognate metal, though either could potentially serve the proposed Lewis acidic role. Subtle differences in the protein structure are implicated in serving as a selectivity filter for Mn(II) or Fe(II).IMPORTANCEThe Irving-Williams series refers to the predicted stabilities of transition metal complexes where the observed general stability for divalent first-row transition metal complexes increase across the row. Acetone carboxylases (ACs) use a coordinated divalent metal at their active site in the catalytic conversion of bicarbonate and acetone to form acetoacetate. Highly homologous ACs discriminate among different divalent metals at their active sites such that variations of the enzyme prefer Mn(II) over Fe(II), defying Irving-Williams-predicted behavior. Defining the determinants that promote metal discrimination within the first-row transition metals is of broad fundamental importance in understanding metal-mediated catalysis and metal catalyst design., Competing Interests: The authors declare no conflict of interest.

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins constitute an adaptive immune system in bacteria and archaea, where immunological memory is retained in the CRISPR locus as short pieces of the intruding nucleic acid, termed spacers. The adaptation to new infections occurs through the integration of a new spacer into the CRISPR array. For immune protection, spacers are transcribed into CRISPR RNAs (crRNA) that are used to guide the effector nuclease of the system in sequence-dependent target cleavage. Spacers originate as a prespacer from either DNA or RNA depending on the CRISPR-Cas system being observed, and the nearly universal Cas proteins, Cas1 and Cas2, insert the prespacer into the CRISPR locus during adaptation in all systems that contain them. The mechanism of site-specific prespacer integration varies across CRISPR classes and types, and distinct differences can even be found within the same subtype. In this review, the current knowledge on the mechanisms of prespacer integration in type II-A CRISPR-Cas systems will be described. Comparisons of the currently characterized type II-A systems show that distinct mechanisms exist within different members of this subtype and are correlated to sequence-specific interactions of Cas proteins and the DNA elements present in the CRISPR array. These observations indicate that nature has fine-tuned the mechanistic details while performing the basic step of DNA integration by Cas proteins, which offers unique advantages to develop Cas1-Cas2-based biotechnology., (© 2022. Springer Nature Switzerland AG.)