Biology subjects

Aphasizheva, I.

Publications and source records attributed to Aphasizheva, I..

4 recordsLinked to original sources

bioRxiv2023

The cryo-EM structure of trypanosome 3-methylcrotonyl-CoAcarboxylase provides mechanistic and dynamic insights into enzymatic function

3-methylcrotonyl-CoA carboxylase (MCC) catalyzes the two-step, biotin-dependent production of 3-methylglutaconyl-CoA, an essential intermediate in leucine catabolism. Given its critical metabolic role, deficiencies in this enzyme associate with organic aciduria, while its overexpression is linked to tumor development. MCC is a dodecameric enzyme composed of six copies of each - and {beta}-subunit. We present the cryo-EM structure of the endogenous MCC holoenzyme from Trypanosoma brucei in its soluble, non-filamentous state at 2.5 [A] resolution. We unambiguously observe the position of biotin, covalently-bound to the BBCP domain of -subunits and occupying a novel binding pocket next to the active site of a neighboring {beta}-subunit dimer. Moreover, flexibility of key residues at the /- and /{beta}-subunit interfaces enables pivoting of -subunit trimers to sequentially approach the otherwise distant active sites for the two steps in MCC catalysis. Our results provide a structural framework to understand the enzymatic mechanism of eukaryotic MCCs and assist drug discovery against trypanosome infections.

molecular biology↗

bioRxiv2022

CryoEM Reveals Oligomeric Isomers of a Multienzyme Complex and Assembly Mechanics

Propionyl-CoA carboxylase (PCC) is a multienzyme complex consisting of up to six -subunits and six {beta}-subunits. Belonging to a metabolic pathway converging on the citric acid cycle, it is present in most forms of life and irregularities in its assembly lead to serious illness in humans, known as propionic acidemia. Here, we report the cryogenic electron microscopy (cryoEM) structures and assembly of different oligomeric isomers of endogenous PCC from the parasitic protozoan Leishmania tarentolae (LtPCC). These structures and their statistical distribution reveal the mechanics of PCC assembly and disassembly at equilibrium. We show that, in solution, endogenous LtPCC {beta}-subunits form stable homohexamers, to which different numbers of -subunits attach. Sorting LtPCC particles into seven classes (i.e., oligomeric formulas 0{beta}6, 1{beta}6, 2{beta}6, 3{beta}6, 4{beta}6, 5{beta}6, 6{beta}6) enables formulation of a model for PCC assembly. Our results suggest how multimerization regulates PCC enzymatic activity and showcase the utility of cryoEM in revealing the statistical mechanics of reaction pathways.

biophysics↗

bioRxiv2022

Discovery, Structure, and Function of Filamentous 3-Methylcrotonyl-CoA Carboxylase

3-methylcrotonyl-CoA carboxylase (MCC) is a biotin-dependent enzyme necessary for leucine catabolism in most organisms. While the crystal structure of recombinant bacterial MCC has been characterized, the structure and potential polymerization of native MCC remain elusive. Here, we discovered that native MCC from Leishmania tarentolae (LtMCC) forms filaments and determined its structure at near-atomic resolution using cryoEM. 6{beta}6 LtMCC dodecamers assemble in a twisted-stacks architecture, manifesting as supramolecular rods extending up to approximately 400 nanometers. LtMCCs in filaments bind biotin but are not covalently biotinylated and lack coenzyme A. Filaments elongate by stacking 6{beta}6 LtMCCs onto the exterior -trimer of the terminal 6{beta}6 dodecamer. This stacking immobilizes the biotin carboxylase domains, sequestering the enzyme in an inactive state within the mitochondrial matrix. Our results support a new model for LtMCC catalysis, termed the dual-swinging-domains model, and cast new light on the functional significance of polymerization in the carboxylase superfamily and beyond.

molecular biology↗

bioRxiv2018

Transcription initiation defines kinetoplast RNA boundaries

Mitochondrial genomes are often transcribed into polycistronic primary RNAs punctuated by tRNAs whose excision defines mature RNA boundaries. Although kinetoplast DNA lacks tRNA genes, it is commonly held that monophosphorylated 5'-ends of functional molecules typify precursor partitioning by an unknown endonuclease. To the contrary, we demonstrate that in Trypanosoma brucei individual mRNAs and rRNAs are independently synthesized as 3' extended precursors. The transcription-defined 5' terminus is converted into monophosphorylated state by the 5' pyrophosphohydrolase complex, termed PPsome, which is activated by RNA editing substrate binding complex (RESC). Most guide RNAs lack PPsome recognition sites and, therefore, remain triphosphorylated. We provide evidence that both 5' pyrophosphate removal and 3' adenylation are essential for mRNA stabilization. Furthermore, we uncover a mechanism by which antisense RNA-controlled 3'-5' exonucleolytic trimming defines mRNA 3'-end. We conclude that mitochondrial mRNAs and rRNAs are transcribed and processed as insulated units irrespective of their genomic location.\n\nSignificanceIt is commonly held that in trypanosomes both mitochondrial DNA strands are transcribed into polycistronic precursors. These primary RNAs are presumably partitioned into individual pre-mRNAs by a \"cryptic\" endonuclease. We challenged the polycistronic transcription/ endonuclease model after revealing precursor processing by 3'-5' degradation. This work demonstrates individual transcription of each gene and mRNA 5'-end definition by the first incorporated nucleotide triphosphate. We have uncovered the stabilizing role of 5' triphosphate to monophosphate conversion and identified a protein complex responsible for this reaction. We have discovered antisense noncoding RNA originating near mRNA 3' end and showed that a duplex formation modulates exonuclease activity to delimit the mature 3' end. Collectively, our findings reveal mechanisms by which transcription defines both mRNA termini.

biochemistry↗