PDBsum entry 4zgj

Lyase

PDB id: 4zgj

Contents

Protein chains

(+ 2 more) 206 a.a.

(+ 2 more) 206 a.a.

Metals

_FE ×8

Waters ×4994

PDB id:

4zgj

Name:

Lyase

Title:

Double mutant h80a/h81a of fe-type nitrile hydratase from comamonas testosteroni ni1

Structure:

Nitrile hydratase alpha subunit. Chain: a, c, e, g, i, k, m, o. Engineered: yes. Mutation: yes. Nitrile hydratase beta subunit. Chain: b, d, f, h, j, l, n, p. Engineered: yes

Source:

Comamonas testosteroni. Organism_taxid: 285. Expressed in: escherichia coli. Expression_system_taxid: 562. Expression_system_taxid: 562

Resolution:

2.00Å R-factor: 0.221 R-free: 0.253

Authors:

R.Wu,S.Martinez,R.Holz,D.Liu

Key ref:

S.Martinez et al. (2015). Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1. J Biol Inorg Chem, 20, 885-894. PubMed id: 26077812 DOI: 10.1007/s00775-015-1273-3

Date:

23-Apr-15 Release date: 01-Jul-15

Protein chains

J9PBS0  (J9PBS0_COMTE) -  Nitrile hydratase alpha subunit from Comamonas testosteroni

Seq: 209 a.a.

Struc: 206 a.a.*

Protein chains

J9PBS1  (J9PBS1_COMTE) -  Nitrile hydratase subunit beta from Comamonas testosteroni

Seq: 206 a.a.

Struc: 206 a.a.*

* PDB and UniProt seqs differ at 5 residue positions (black crosses)

Enzyme reactions

• Enzyme class: Chains A, B, C, D, E, F, G, H, I, J, K, L, M, N, O, P: E.C.4.2.1.84 - nitrile hydratase.
• Reaction: an aliphatic primary amide = an aliphatic nitrile + H2O

Molecule diagrams generated from .mol files obtained from the KEGG ftp site

Key reference

DOI no: 10.1007/s00775-015-1273-3

J Biol Inorg Chem 20:885-894 (2015)

PubMed id: 26077812

Analyzing the catalytic role of active site residues in the Fe-type nitrile hydratase from Comamonas testosteroni Ni1.

S.Martinez, R.Wu, K.Krzywda, V.Opalka, H.Chan, D.Liu, R.C.Holz.

ABSTRACT

A strictly conserved active site arginine residue (αR157) and two histidine residues (αH80 and αH81) located near the active site of the Fe-type nitrile hydratase from Comamonas testosteroni Ni1 (CtNHase), were mutated. These mutant enzymes were examined for their ability to bind iron and hydrate acrylonitrile. For the αR157A mutant, the residual activity (k cat = 10 ± 2 s(-1)) accounts for less than 1 % of the wild-type activity (k cat = 1100 ± 30 s(-1)) while the K m value is nearly unchanged at 205 ± 10 mM. On the other hand, mutation of the active site pocket αH80 and αH81 residues to alanine resulted in enzymes with k cat values of 220 ± 40 and 77 ± 13 s(-1), respectively, and K m values of 187 ± 11 and 179 ± 18 mM. The double mutant (αH80A/αH81A) was also prepared and provided an enzyme with a k cat value of 132 ± 3 s(-1) and a K m value of 213 ± 61 mM. These data indicate that all three residues are catalytically important, but not essential. X-ray crystal structures of the αH80A/αH81A, αH80W/αH81W, and αR157A mutant CtNHase enzymes were solved to 2.0, 2.8, and 2.5 Å resolutions, respectively. In each mutant enzyme, hydrogen-bonding interactions crucial for the catalytic function of the αCys(104)-SOH ligand are disrupted. Disruption of these hydrogen bonding interactions likely alters the nucleophilicity of the sulfenic acid oxygen and the Lewis acidity of the active site Fe(III) ion.