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

Abstract

L-Amino acid deaminase from Proteus myxofaciens (Pma-LAAD) is a membrane flavoenzyme that catalyzes the deamination of neutral and aromatic L-amino acids into ␣-keto acids and ammonia. PmaLAAD does not use dioxygen to re-oxidize reduced FADH 2 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 ␣؉␤ subdomain placed close to the putative transmembrane ␣-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.A variety of enzymes has been used to prepare chiral pharmaceutical and agricultural compounds containing enantiomeric amine or amino acid groups. Among these are aminotransferases (EC 2.6.4.X), lipases (EC 3.1.1.X), amine oxidases (EC 1.4.3.22), amino acid dehydrogenases (EC 1.4.1.X), and amino acid oxidases (EC 1.4.3.X) (1, 2).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 reduction. The second step iteratively converts the imino acid produced (from the D-amino acid) back into a DL-mixture to obtain the full resolution of the racemic mixture into the L-enantiomer (1). This approach requires stable recombinant DAAOs possessing wide substrate specificity as well as variants engineered to act on synthetic amino acids (3). Amino acid oxidases with reverse stereoselectivity are also well known flavooxidases, mainly produced by snakes or by microorganisms. In particular, L-amino acid oxidases (LAAO, EC 1.4.3.2) catalyze the stereoselective oxidative deamination of L-amino acids into the corresponding ␣-keto acids and ammonia; the re-oxidation of FADH 2 by dioxygen then generates H 2 O 2 (4). These flavoenzymes catalyze an irreversible reaction (differently from aminotransferases) and do not require a specific step of cofactor regeneration, as otherwise required by the NAD-dependent dehydrogenases. However, because of the problems associated with overexpression of snake venom LAAOs in recombinant hosts and the limited substrate acceptance of the microbial counterparts, no appropriate LAAOs for biocatalysis are available (4).L-Amino acid deaminases (LAADs) represent a suitable alternative to LAAOs. LAAD (first identified in the genera Proteus, Providencia, and Morganella) catalyzes the deamination of the L-isomer of amino acids, yielding the corresponding ␣-keto acids and ammonia without any evide...

Deracemization and Stereoinversion of α‐Amino Acids by l‐Amino Acid Deaminase

“…Overexpressed in E. coli cells, the enzyme was easily purified by a single chromatographic step, recovered full activity when incubated with membranes and, notably, did not require exogeneous FAD for maximal activity. The activity of PmaLAAD is highest at pH 7.5 and at 50 °C; indeed, the enzyme is quite stable . PmaLAAD prefers bulky and hydrophobic substrates, such as l ‐Phe, l ‐Leu, l ‐Met, and l ‐Trp, followed by the polar amino acid l ‐Cys.…”

“…Here, a suitable alternative is represented by l ‐amino acid deaminase (LAAD). This membrane‐associated, FAD‐containing enzyme catalyzes the O 2 ‐dependent deamination of l ‐amino acids similarly to LAAO (and DAAO on the opposite enantiomer), in this case also yielding the corresponding α‐keto acids and ammonia; however, the electrons are transferred from the reduced cofactor to a cytochrome‐b‐like acceptor without producing any hydrogen peroxide (see Scheme A) …”

“…When LAADs are separated from membranes, O 2 consumption decreases dramatically: the reduced FAD cofactor of LAAD from Proteus myxofaciens reacts very slowly with dioxygen with a rate constant of 0.08 s −1 at air saturation …”

“…LAADs are monomeric enzymes showing a low sequence identity with canonical homodimeric LAAOs or DAAOs . LAADs have been identified in Proteus bacteria only.…”

“…PmaLAAD prefers bulky and hydrophobic substrates, such as l ‐Phe, l ‐Leu, l ‐Met, and l ‐Trp, followed by the polar amino acid l ‐Cys. It also shows the ability to deaminate small apolar or charged amino acids at a low rate . Furthermore, PmaLAAD was also reported to be active on synthetic and biotechnologically relevant amino acids, such as l ‐3,4‐dihydroxyphenylalanine ( l ‐DOPA) and substituted alanines …”