Structure-Function Relationships in l-Amino Acid Deaminase, a Flavoprotein Belonging to a Novel Class of Biotechnologically Relevant Enzymes

Paolo Motta,Gianluca Molla,Loredano Pollegioni,Marco Nardini

doi:10.1074/jbc.m115.703819

Abstract

l-Amino acid deaminase from Proteus myxofaciens (PmaLAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic l-amino acids into α-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH2 and thus does not produce hydrogen peroxide; instead, it uses a cytochrome b-like protein as an electron acceptor. Although the overall fold of this enzyme resembles that of known amine or amino acid oxidases, it shows the following specific structural features: an additional novel α+β subdomain placed close to the putative transmembrane α-helix and to the active-site entrance; an FAD isoalloxazine ring exposed to solvent; and a large and accessible active site suitable to bind large hydrophobic substrates. In addition, PmaLAAD requires substrate-induced conformational changes of part of the active site, particularly in Arg-316 and Phe-318, to achieve the correct geometry for catalysis. These studies are expected to pave the way for rationally improving the versatility of this flavoenzyme, which is critical for biocatalysis of enantiomerically pure amino acids.

Highlights

The deracemization of a racemic amino acid to obtain the L-configuration was achieved by using a stereoselective Damino acid oxidase (DAAO,5 EC 1.4.3.3) followed by chemical
L-Amino acid deaminases (LAADs), L-amino acid deaminase from P. myxofaciens; PmaLAAD-00C, C-terminal His-tagged PmaLAAD; PmaLAAD-00N, N-terminal His-tagged PmaLAAD; PmaLAAD-01N, truncated PmaLAAD-00N starting from Met-28; PmaLAAD-02N, truncated PmaLAAD-00N starting from Ala-50; Sbd, substrate binding domain; PDB, Protein Data Bank
Thawing, diluting (5-fold) in 20 mM triethanolamine-HCl, pH 7.5, and centrifuging the membrane fraction stored at Ϫ80 °C resulted in a phase separation; the lower, denser phase contained about half of the PmaLAAD of the sample and showed a further Ϸ2.5-fold increase in specific activity, reaching 2.9 units/mg of protein, with an overall yield of 33% (Fig. 2)

Summary

Experimental Procedures

Cloning of PmaLAAD Wild-type and Variants—The gene encoding for LAAD from P. myxofaciens (newly classified as Cosenzaea myxofaciens) [8] cloned into the NdeI and SalI restriction sites of the pET21a expression plasmid (Merck) was a generous gift from Wolfgang Kroutil (University of Graz, Austria). Flavin reduction was carried out by adding 25 mM (final concentrations) of the substrate L-Phe to samples containing Ϸ15 ␮M enzyme; reduction of the cofactor for PmaLAAD-00N variant was performed both in the absence and in the presence of E. coli membranes. Binding experiments of carboxylic acids and sulfite to PmaLAAD-00N and -01N variants were performed by adding small volumes (5–50 ␮l) of concentrated stock solutions (0.1 mM to 1 M) to samples containing 0.8 ml of Ϸ15 ␮M enzyme and following the changes in the visible absorbance spectrum of the flavin cofactor [13, 14]. Poor electron density is present at the 28 –31 and 337–342 regions and at the C terminus (residue 474) of both A and B chains

Water molecules

Results

Relative specific activity of PmaLAAD on different substrates

Amino acid

Aligned No of PDB code Chain Z score

Discussion