Dataset Information

Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

ABSTRACT: Cardiac senescence is a typical chronic frailty condition in the elderly population, and cellular aging is often associated with oxidative stress. The mitochondrial-membrane flavoenzyme monoamine oxidase A (MAO A) catalyzes the oxidative deamination of neurotransmitters, and its expression increases in aged hearts. We produced recombinant human MAO A variants at Lys305 that play a key role in O₂ reactivity leading to H₂O₂ production. The K305Q variant is as active as the wild-type enzyme, whereas K305M and K305S have 200-fold and 100-fold lower k_cat values and similar K_m. Under anaerobic conditions, K305M MAO A was normally reduced by substrate, whereas reoxidation by O₂ was much slower but could be accomplished by quinone electron acceptors. When overexpressed in cardiomyoblasts by adenoviral vectors, the K305M variant showed enzymatic turnover similar to that of the wild-type but displayed decreased ROS levels and senescence markers. These results might translate into pharmacological treatments as MAO inhibitors may attenuate cardiomyocytes aging.

SUBMITTER: Iacovino LG

PROVIDER: S-EPMC8009472 | biostudies-literature | 2020 Jul

REPOSITORIES: biostudies-literature

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Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

Iacovino Luca Giacinto LG Manzella Nicola N Resta Jessica J Vanoni Maria Antonietta MA Rotilio Laura L Pisani Leonardo L Edmondson Dale Edward DE Parini Angelo A Mattevi Andrea A Mialet-Perez Jeanne J Binda Claudia C

ACS chemical biology 20200630 7

Cardiac senescence is a typical chronic frailty condition in the elderly population, and cellular aging is often associated with oxidative stress. The mitochondrial-membrane flavoenzyme monoamine oxidase A (MAO A) catalyzes the oxidative deamination of neurotransmitters, and its expression increases in aged hearts. We produced recombinant human MAO A variants at Lys305 that play a key role in O2 reactivity leading to H2O2 production. The K305Q variant is as activ ...[more]

PMID: 32589395

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Project description:Glucose oxidase (GOx) is an enzymatic workhorse used in the food and wine industries to combat microbial contamination, to produce wines with lowered alcohol content, as the recognition element in amperometric glucose sensors, and as an anodic catalyst in biofuel cells. It is naturally produced by several species of fungi, and genetic variants are known to differ considerably in both stability and activity. Two of the more widely studied glucose oxidases come from the species Aspergillus niger (A. niger) and Penicillium amagasakiense (P. amag.), which have both had their respective genes isolated and sequenced. GOx from A. niger is known to be more stable than GOx from P. amag., while GOx from P. amag. has a six-fold superior substrate affinity (K(M)) and nearly four-fold greater catalytic rate (k(cat)). Here we sought to combine genetic elements from these two varieties to produce an enzyme displaying both superior catalytic capacity and stability. A comparison of the genes from the two organisms revealed 17 residues that differ between their active sites and cofactor binding regions. Fifteen of these residues in a parental A. niger GOx were altered to either mirror the corresponding residues in P. amag. GOx, or mutated into all possible amino acids via saturation mutagenesis. Ultimately, four mutants were identified with significantly improved catalytic activity. A single point mutation from threonine to serine at amino acid 132 (mutant T132S, numbering includes leader peptide) led to a three-fold improvement in k(cat) at the expense of a 3% loss of substrate affinity (increase in apparent K(M) for glucose) resulting in a specify constant (k(cat)/K(M)) of 23.8 (mM(-1) · s(-1)) compared to 8.39 for the parental (A. niger) GOx and 170 for the P. amag. GOx. Three other mutant enzymes were also identified that had improvements in overall catalysis: V42Y, and the double mutants T132S/T56V and T132S/V42Y, with specificity constants of 31.5, 32.2, and 31.8 mM(-1) · s(-1), respectively. The thermal stability of these mutants was also measured and showed moderate improvement over the parental strain.

Dataset Information

Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

Publications

Rational Redesign of Monoamine Oxidase A into a Dehydrogenase to Probe ROS in Cardiac Aging.

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