Crystal Structure of a Novel FAD-, FMN-, and ATP-containing l-Proline Dehydrogenase Complex from Pyrococcus horikoshii

Abstract

Two novel types of dye-linked L-proline dehydrogenase complex (PDH1 and PDH2) were found in a hyperthermophilic archaeon, Pyrococcus horikoshii OT3. Here we report the first crystal structure of PDH1, which is a heterooctameric complex (␣␤) 4 containing three different cofactors: FAD, FMN, and ATP. The structure was determined by x-ray crystallography to a resolution of 2.86 Å. The structure of the ␤ subunit, which is an L-proline dehydrogenase catalytic component containing FAD as a cofactor, was similar to that of monomeric sarcosine oxidase. On the other hand, the ␣ subunit possessed a unique structure composed of a classical dinucleotide fold domain with ATP, a central domain, an N-terminal domain, and a Cys-clustered domain. Serving as a third cofactor, FMN was located at the interface between the ␣ and ␤ subunits in a novel configuration. The observed structure suggests that FAD and FMN are incorporated into an electron transfer system, with electrons passing from the former to the latter. The function of ATP is unknown, but it may play a regulatory role. Although the structure of the ␣ subunit differs from that of the ␤ subunit, except for the presence of an analogous dinucleotide domain with a different cofactor, the structural characteristics of PDH1 suggest that each represents a divergent enzyme that arose from a common ancestral flavoenzyme and that they eventually formed a complex to gain a new function. The structural characteristics described here reveal the PDH1 complex to be a unique diflavin dehydrogenase containing a novel electron transfer system. Dye-linked dehydrogenases catalyze the oxidation of various amino acids, organic acids, amines, and alcohols in the presence of an artificial electron acceptor such as ferricyanide or 2,6-dichloroindophenol (DCIP). 1 We have identified several novel dye-linked dehydrogenases among the hyperthermophilic archaea, including D-proline dehydrogenase (1) and Lproline dehydrogenase (PDH) (2, 3), which catalyzes the dehydrogenation from L-prroline to ⌬ 1 -pyrroline-5-carboxylate (P5C). The gene sequence and primary structure of the first PDH isolated from a hyperthermophilic archaeon, Thermococcus profundus DSM9503, have been determined (2, 3). The complete gene is formed by an operon comprised of four genes, pdhA, pdhB, pdhF and pdhX, in a tandem arrangement in the order pdhA-F-X-B. The purified enzyme has a native molecular mass of 120 kDa and forms an unusual ␣␤␥␦ heterotetrameric complex (␣ (54 kDa), ␤ (43 kDa), ␥ (19 kDa), and ␦ (11 kDa)) encoded by pdhA, pdhB, pdhF, and pdhX, respectively. In addition, functional analysis of each subunit has shown that the ␣ and ␤ subunits possess NADH and L-proline dehydrogenase activities, respectively (3). Because this multifunctional dye-linked dehydrogenase complex appears to representative of a new enzyme group (3), we have been seeking others by screening dye-linked dehydrogenases expressed in hyperthermophilic archaea. So far, we have identified two different types of dye-linked PDH in the hyperthermophilic ar...

Structure and Kinetics of Monofunctional Proline Dehydrogenase from Thermus thermophilus

“…For example, PDH1 is an (␣␤) 4 hetero-octameric complex with the ␤ subunit binding an FAD cofactor and exhibiting PRODH activity (6). The crystal structure of PDH1 shows that the FAD-binding domain has a Rossmann dinucleotide-binding fold similar to that of monomeric sarcosine oxidase, which is a member of the glutathione reductase family (6). As expected for a Rossmann fold protein, the FAD of PDH1 is highly extended, and the pyrophosphate interacts with a glycine-rich loop and associated conserved water molecule (45).…”

“…Studies of the bacterial enzymes have shown that PRODH is an FAD-dependent enzyme with a (␤␣) 8 barrel catalytic core (3,4), and P5CDH is an NAD ϩ -dependent Rossmann fold enzyme featuring a nucleophilic Cys (5). These enzymes are unrelated in sequence and structure to hyperthermophilic archaeal proline catabolic enzymes, which appear in unique hetero-tetrameric and hetero-octameric complexes (6).…”

Identification and Characterization of d-Hydroxyproline Dehydrogenase and Δ1-Pyrroline-4-hydroxy-2-carboxylate Deaminase Involved in Novel l-Hydroxyproline Metabolism of Bacteria

“…First, L-PDH of Thermococcus profundus (Tp/L-PDH) forms a complex consisting of four different subunits with molecular masses of 54 (␣), 42 (␤), 19 (␥), and 11 kDa (␦), respectively, with a heterotetrameric structure of ␣␤␥␦ containing two FADs (in ␣-and ␤-subunits) (8). Second, L-PDH isozyme 1 of Pyrococcus horikoshii (Ph/L-PDH1) consists of two different subunits with molecular masses of 56 (␣) and 43 kDa (␤) with a heterooctameric structure of ␣ 4 ␤ 4 containing one FAD (␤), one FMN (between ␣-and ␤-subunits), and one ATP (␣) (9,10). P. horikoshii also possesses L-PDH isozyme 2 (Ph/L-PDH2), which is similar to Tp/L-PDH.…”

Crystal Structure of Novel Dye-linked l-Proline Dehydrogenase from Hyperthermophilic Archaeon Aeropyrum pernix

“…The main chain coordinates of the ApeLPDH monomer were essentially the same as those of the PDH1 ␤-subunit (PDH1␤) (Protein Data Bank code 1Y56-B) (11). Superposition of PDH1␤ onto ApeLPDH yielded a root mean square deviation (r.m.s.d.)…”

“…The PDH1 hetero-octamer reportedly assembles as a tetramer of the ␣␤-heterodimer, within which one (␣␤) 2 block generated by an intermolecular disulfide bridge (formed between the two ␣-subunits) binds with another at the back to form the (␣␤) 4 -hetero-octamer (Fig. 6A) (11). Within the hetero-octamer, ␤-subunits interact only with ␣-subunits, and one molecule of FMN is located at the interface between the ␣-and ␤-subunits (Fig.…”