Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1

Mariya S. Adachi, Alexander B. Taylor, P. John Hart, Paul F Fitzpatrick

Research output: Contribution to journalArticle

6 Citations (Scopus)

Abstract

The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N 1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N 1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k cat/K O2 value changes very little upon mutation with N 1-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k cat/K M-pH profiles with N 1-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK a values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK a as wild-type Fms1, about ∼7.4; this pK a is assigned to the substrate N4. The k cat/K O2-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK a of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k cat value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure (Graph Presented).

Original languageEnglish (US)
Pages (from-to)4888-4897
Number of pages10
JournalBiochemistry
Volume51
Issue number24
DOIs
StatePublished - Jun 19 2012

Fingerprint

Yeast
Yeasts
Spermine
Cats
Substrates
Enzymes
Catalytic Domain
Mutation
Amines
Hydrogen
Rate constants
Hydrogen bonds
Flavoproteins
Spermidine
Polyamines
Catalysis
Saccharomyces cerevisiae
Oxidoreductases
polyamine oxidase
Oxidation

ASJC Scopus subject areas

  • Biochemistry

Cite this

Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1. / Adachi, Mariya S.; Taylor, Alexander B.; Hart, P. John; Fitzpatrick, Paul F.

In: Biochemistry, Vol. 51, No. 24, 19.06.2012, p. 4888-4897.

Research output: Contribution to journalArticle

@article{d56a1b18259442d291640c41c2dbc641,
title = "Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1",
abstract = "The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N 1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N 1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k cat/K O2 value changes very little upon mutation with N 1-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k cat/K M-pH profiles with N 1-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK a values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK a as wild-type Fms1, about ∼7.4; this pK a is assigned to the substrate N4. The k cat/K O2-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK a of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k cat value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 {\AA}. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure (Graph Presented).",
author = "Adachi, {Mariya S.} and Taylor, {Alexander B.} and Hart, {P. John} and Fitzpatrick, {Paul F}",
year = "2012",
month = "6",
day = "19",
doi = "10.1021/bi300517s",
language = "English (US)",
volume = "51",
pages = "4888--4897",
journal = "Biochemistry",
issn = "0006-2960",
publisher = "American Chemical Society",
number = "24",

}

TY - JOUR

T1 - Mechanistic and structural analyses of the role of His67 in the yeast polyamine oxidase Fms1

AU - Adachi, Mariya S.

AU - Taylor, Alexander B.

AU - Hart, P. John

AU - Fitzpatrick, Paul F

PY - 2012/6/19

Y1 - 2012/6/19

N2 - The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N 1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N 1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k cat/K O2 value changes very little upon mutation with N 1-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k cat/K M-pH profiles with N 1-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK a values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK a as wild-type Fms1, about ∼7.4; this pK a is assigned to the substrate N4. The k cat/K O2-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK a of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k cat value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure (Graph Presented).

AB - The flavoprotein oxidase Fms1 from Saccharomyces cerevisiae catalyzes the oxidation of spermine and N 1-acetylspermine to spermidine and 3-aminopropanal or N-acetyl-3-aminopropanal. Within the active site of Fms1, His67 is positioned to form hydrogen bonds with the polyamine substrate. This residue is also conserved in other polyamine oxidases. The catalytic properties of H67Q, H67N, and H67A Fms1 have been characterized to evaluate the role of this residue in catalysis. With both spermine and N 1-acetylspermine as the amine substrate, the value of the first-order rate constant for flavin reduction decreases 2-3 orders of magnitude, with the H67Q mutation having the smallest effect and H67N the largest. The k cat/K O2 value changes very little upon mutation with N 1-acetylspermine as the amine substrate and decreases only an order of magnitude with spermine. The k cat/K M-pH profiles with N 1-acetylspermine are bell-shaped for all the mutants; the similarity to the profile of the wild-type enzyme rules out His67 as being responsible for either of the pK a values. The pH profiles for the rate constant for flavin reduction for all the mutant enzymes similarly show the same pK a as wild-type Fms1, about ∼7.4; this pK a is assigned to the substrate N4. The k cat/K O2-pH profiles for wild-type Fms1 and the H67A enzyme both show a pK a of about ∼6.9; this suggests His67 is not responsible for this pH behavior. With the H67Q, H67N, and H67A enzymes the k cat value decreases when a single residue is protonated, as is the case with the wild-type enzyme. The structure of H67Q Fms1 has been determined at a resolution of 2.4 Å. The structure shows that the mutation disrupts a hydrogen bond network in the active site, suggesting that His67 is important both for direct interactions with the substrate and to maintain the overall active site structure (Graph Presented).

UR - http://www.scopus.com/inward/record.url?scp=84862537083&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=84862537083&partnerID=8YFLogxK

U2 - 10.1021/bi300517s

DO - 10.1021/bi300517s

M3 - Article

C2 - 22642831

AN - SCOPUS:84862537083

VL - 51

SP - 4888

EP - 4897

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 24

ER -