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2'- O -ribose methylation of the first transcribed base (adenine or A 1 in SARS-CoV-2) of viral RNA mimics the host RNAs and subverts the innate immune response. How nsp16, with its obligate partner nsp10, assembles on the 5'-end of SARS-CoV-2 mRNA to methylate the A 1 has not been fully understood. We present a ∼ 2.4 Å crystal structure of the heterotetrameric complex formed by the cooperative assembly of two nsp16/nsp10 heterodimers with one 10-mer Cap-1 RNA (product) bound to each. An aromatic zipper-like motif in nsp16 and the N-terminal regions of nsp10 and nsp16 orchestrate an oligomeric assembly for efficient methylation. The front catalytic pocket of nsp16 stabilizes the upstream portion of the RNA while the downstream RNA remains unresolved, likely due to its flexibility. An inverted nsp16 dimer extends the positively charged surface area for longer RNA to influence the catalysis. Additionally, a non-specific nucleotide-binding pocket on the backside of nsp16 plays a critical role in catalysis, further contributing to its enzymatic activity.

l-cysteine, a primary building block of mycothiol, plays an essential role in the defense mechanism of Mycobacterium tuberculosis (Mtb). However, it is unclear how Mtb regulates cysteine biosynthesis as no study has reported the cysteine regulatory complex (CRC) in Mtb. Serine acetyltransferase (SAT) and cysteine synthase (CS) interact to form CRC. Although MtCS has been characterized well, minimal information is available on MtSAT, which synthesizes, O-acetylserine (OAS), the precursor of cysteine. This study fills the gap and provides experimental evidence for the presence of MtCRC and a non-canonical multi-oligomeric MtSAT. We employed multiple analytical methods to characterize the oligomeric and kinetic properties of MtSAT and MtCRC. Results show that MtSAT, lacking >75 N-terminal amino acids exists in three different assembly states; trimer, hexamer, and dodecamer, compared to the single hexameric state of SAT of other bacteria. While hexamers display the highest catalytic turnover, the trimer is the least active. The predominance of trimers at low physiologically relevant concentrations suggests that MtSAT displays the lowest catalytic potential known. Further, the catalytic potential of MtSAT is also significantly reduced in CRC state, in contrast to enhanced activity of SAT in CRC of other organisms. Our study provides insights into multi-oligomeric MtSAT with reduced catalytic potential and demonstrates that both MtSAT and MtCS of Mycobacterium interact to form CRC, although with altered catalytic properties. We discuss our results in light of the altered biochemistry of the last step of canonical sulfate-dependent cysteine biosynthesis of Mycobacterium., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)