SufB intein splicing inMycobacterium tuberculosisis influenced by two remote conserved N-extein Histidines

Abstract

Inteins are auto-processing domains that implement a multi-step biochemical reaction termed protein splicing, marked by cleavage and formation of peptide bonds. They excise from a precursor protein, generating a functional protein via covalent bonding of flanking exteins. We report the kinetic study of splicing and cleavage reaction in a [Fe-S] cluster assembly protein SufB from Mycobacterium tuberculosis. Although it follows a canonical intein splicing pathway, distinct features are added by extein residues present in the active site. Sequence analysis identified two conserved histidines in the N-extein region; His-5 and His-38. Kinetic analyses of His-5Ala and His-38Ala SufB mutants exhibited significant reductions in splicing and cleavage rates relative to the SufB wild-type precursor protein. Structural analysis and molecular dynamics simulations suggested that Mtu SufB displays a unique mechanism where two remote histidines work concurrently to facilitate N- cleavage reaction. His-5, which is exposed outside, because of the random push of water molecules forces His-38 towards the N-cleavage site. Thus, His-5 stabilizes the position of His-38 which in turn activates N-S acyl shift via direct interaction with catalytic Cys1. Understanding intein~extein partnership in an essential mycobacterial protein may diversify into intein-based applied research along with the development of pathogen-specific novel antimicrobials.

Gold Nanoparticles Augment N-Terminal Cleavage and Splicing Reactions in Mycobacterium tuberculosis SufB

“…The identity of splicing and cleavage products were confirmed by MALDI TOF/TOF Mass spectrometry and the results were submitted to the ProteomeXchange Consortium via the PRIDE with data ID PXD015199 (Submission details are provided in the Supplementary Material (Page S7 ).) ( Panda et al, 2021 ).…”

“…The plasmids used in this study are previously engineered, and the details are mentioned elsewhere ( Panda et al, 2021 ). Mtu SufB wild type, splicing inactive double mutant (C1A/N359A) SufB (negative control), and SufB intein all carrying N-terminal 6X (His) tag were overexpressed in BL21 (DE3) E. coli cells via IPTG (sigma 367-93-1) induction.…”

“…The structure of Mtu SufB precursor has been depicted in Figure 6 , where the amino acid sequence, 251–254, has been shown as EGCL ( Panda et al, 2021 ). According to the previous model ( Dearden et al, 2013 ; Dearden, 2014 ), we have selected the active site (Gly-Cys) with capping of the –CH3 (carboxylic terminal) and –CONH 2 group (amide terminal), as shown in Figure 6 .…”

“… 3D structure of Mtu full-length SufB precursor is obtained from Model Archive [( https://www.modelarchive.org/doi/10.5452/ma-x807d , Access code-6pmXRNkvwR] ( Panda et al, 2021 ). EGCL represents the amino acid residues from 251 to 254.…”