Crystal structure of YagE, a putative DHDPS‐like protein from <i>Escherichia coli K12</i>

Abstract

The incorporation of genetic material from other organisms into bacterial genomes potentiates novel forms of drug resistance or pathogenicity. One method of incorporation is through infection by a bacteriophage, which enters the lysogenic life cycle. Excision of the phage genome as a result of induction may include some of the host's genome, and subsequent infection and lysogeny in other bacteria would incorporate this nonphage DNA into these hosts. Loss of ability to excise itself from the host leads to the permanent addition in the host genome of the phage genes and other genetic material that may have accumulated during previous lysogenic phases. In this project, targets were chosen initially from proteins encoded by prophages present in E. coli K12 based on possible similarities to known virulence factors from pathogenic bacteria, whose three-dimensional structures are unknown. One such target was the yagE gene located in the E. coli K12 genome that is part of prophage CP4-61 encoding a 33-kDa putative dihydrodipicolinate synthase (DHDPS)-like protein (UniProtKB/Swiss-prot: P75682). The DHDPS-like domain belongs to the N-acetyl neuraminate lyase (NAL) subfamily2 containing an eightfold α/β barrel (TIM barrel) with a small C-terminal α-helical domain (Inter pro: IPR005263). The DHDPS domain is present in a wide variety of enzymes. For example, NAL, which is involved in sialic acid metabolism, trans-o-hydroxybenzylidine pyruvate hydratase aldolase (HBPHA) in naphthalene degradation, D-4-deoxy-5-oxoglucarate dehydratase (DOGDH) in glucarate metabolism, 2-keta-3-deoxy gluconate aldolase (KDGA) in the Entner-Duodoroff pathway, and dihydrodipicolinate synthase (DHDPS) in lysine biosynthesis. All these enzymes contain the DHDPS-like domain and the small C-terminal α-helical region [Fig. 1(A)]. Comparison of YagE structure and sequence with related proteins (A) cartoon representation of YagE monomer. (B) Cartoon representation of YagE tetramer showing the twofold symmetry axes between monomers. Chain A is colored green, Chain B, cyan, Chain C, magenta, and Chain D, yellow. The third axis is perpendicular to the page. (C) Superposition of YagE monomer with EcDHDSP, BaDHDPS, EcNAL, and SsKDGA monomers. (D) Sequence alignment of YagE with EcDHDPS (1XKY), BaDHDPS (2ATS), EcNAL (1NAL), and SsKDGA (1W3I) figure generated using ESPript.3 Of all the TIM barrel protein folds described so far, the most common is the eightfold α/β barrel and all family members are enzymes except narbonin.4 Proteins that contain DHDPS-like domains catalyze a wide variety of reactions, all of which involve formation of a Schiff's base intermediate.5 A lysine residue in β-strand 6 on the floor of the active site region forms a Schiff's base with one of the substrates,6 which in most of these enzymes is pyruvate. Substrate promiscuity is often reported for some of its members like KDG aldolase.7 The fold is very amenable to directed evolution studies as seen in the case of NAL, where DHDPS activity can be gained by a single point mutation (L142R) while NAL activity is retained.2 Crystal structures of proteins with a DHDPS-like domain have been determined from several organisms. Some of the available PDB models include DHDPS from E. coli8 (EcDHDPS, PDB: 2ATS), Thermotoga maritima9 (PDB ID code: 1O5K), Bacillus anthracis10 (BaDHDPS, PDB ID code: 1XKY), Nicotiana sylvestris11 (PDB ID code), NAL from E. coli12 (EcNAL, PDB ID code: 1NAL), Haemophilus influenza13 (PDB ID code: 1F6K), and KDG aldolase from Sulfolobus solfataricus (SsKDGA, PDB ID code: 1W3I). The sequence similarity of YagE to any of these structures is very low (≤30%). Thus the availability of a crystal structure of YagE would help address the question of the functionality and structural relatedness of YagE in relation to the DHDPS-like domain proteins. The initial clone of the yagE gene was obtained from Genobase, Tokyo, in a pCA24N vector. Subsequently, using 5′-TCACGGTACCCAAGGAGATCTCATGCCG as forwar