The MenH module

The PHYLLO MenH module comprises 302 amino acid residues ranging from the residue position 1.414 to the position 1.715 (Fig. 10). Blast searches with this module returned hits with minimum e-value of 4e-35 with the MenH proteins of eubacteria. In general, these proteins display low levels of sequence identity (Table 6) with PHYLLO, as also observed for the MenD and MenC eubacterial homologs. In addition to MenH proteins, BLAST searches also retrieved hits with several other proteins having the so-called α/β

hydrolase fold. This was confirmed in a search against the CDD returning hits with an e-value 4e-18 with deposited hydrolases and esterases domains, as well as with many subgroups of the α/β hydrolase fold. This superfamily comprises as many different members as proteases, lipases, dehalogenases, peroxidases, epoxide hydrolases and esterases (Nardini and Dijkstra, 1999). This latter function is consistent with the thioesterase activity of MenH proteins.

Table 6. Conservation levels of the MenH module of PHYLLO. The identity (ID) and similarity (Sim) levels of the MenH module of PHYLLO with representative eubacterial MenH proteins are shown. GenBank accession numbers (accession) of proteins are indicated.

MenH of accession ID Sim Trichodesmium erythraeum ZP_00071896 27% 48% Listeria innocua CAC97013 27% 43% Nostoc punctiforme ZP_00112005 26% 47% Chlorobium tepidum AAM73065 26% 44% Nostoc sp. PCC 7120 BAB74452 25% 44% Bacillus subtilis CAB15059 23% 39% Escherichia coli AAC75323 20% 35%

The α/β hydrolase fold has been described as consisting of parallel, eight-stranded β

sheet surrounded on both sides by α helices (Fig. 16), providing a stable scaffold for the active sites of a wide variety of enzymes (Ollis et al., 1992; Nardini and Dijkstra, 1999). The catalytic residues always constitute a highly conserved triad: a nucleophile (serine, cysteine or aspartate) positioned after strand β5, an acidic residue almost always positioned after strand

PHYLLO

PHYLLOMenHMenHmodulemodule, 302 , 302 amino acidsamino acids

Proposed catalytic triad: S100 D226 H261

H261 H261 S100 S100 D226 D226

Figure 16. The proposed catalytic triad for the Arabidopsis MenH module of PHYLLO. The secondary structure of the canonical α/β hydrolase fold (according to Ollis et al., 1992) is showed above. The α helices and β strands are represented by white cylinders and grey arrows, respectively. The location of the proposed catalytic triad S100, H261 and D226 for the PHYLLO MenH is indicated by arrows and black dots in the upper cartoon and by doted lines in the schematic structure of the MenH module displayed at the lower part of the figure.

β7 and an absolutely conserved histidine following the last β strand (Ollis et al., 1992; Nardini and Dijkstra, 1999). In the Arabidopsis MenH module of PHYLLO such highly conserved residues are observed in the relative position of the protein structure. We propose that the nucleophile S1455, the acidic D1639 and the invariant H1674 are the three residues involved in catalysis in the Arabidopsis MenH module of PHYLLO, as well as in the other MenH proteins (Fig. 16; Fig. 17).

* 20 * 40 * 60 * E. coli : ---MILHAQAKHGKPGLPWLVFLHGFSGDCHEWQEVGEAFADYS-RLYVDLPGHGGSAAIS--- : 57 Nostoc : ---MILKKYKFNYFLNGNTNKPLIIFLHGFIGNIHEFNEAIKLLSEDFCYLTLDLPGHGKTEVLG---G : 63 Listeria : ---MLVNGQHYHLINDISGEKPVLLMLHGFTGSSETFQDSISLLKEHFSIIAPDLLGHGKTSCPK---E : 63 Trichodesm : ---MFNGKNYKFNYSLSQKNNKTIILLLHGFMGSSNDFIEIIPELSKKFCCLTVDLPGHGKTRVFD---S : 64 AtMenH : ---DGFSHFIRVHDVGENAEGSVALFLHGFLGTGEEWIPIMTGISGSARCISVDIPGHGRSRVQSHASETQT : 69 80 * 100 * 120 * 140 * E. coli : --VDGFDDVTDLLRKTLVSYNILDFWLVGYSLGGRVAMMAACQGLAGLCGVIVEGGHPGLQNAEQRAERQRSDRQ : 130 Nostoc : DEYYQMESIAQAIINLLDELRIDKCHLIGYSMGGRLALYLTLHFPQRFIKVVLESACPGLATEVERLERIRRDAQ : 138 Listeria : VARYSIENICDDLASILHQLKIDSCFVLGYSMGGRVATAFAAKYKELVRGLVLVSSSPGLRDEKARASRISADNR : 138 Trichodesm : EKHYNMHNTATALIGLLDNLNIEKCYLFGYSMGGRLALYLGINFPTRFEKIILESASPGLKSKAERSLRCQSDFQ : 139 AtMenH : SPTFSMEMIAEALYKLIEQITPGKVTIVGYSMGARIALYMALRFSNKIEGAVVVSGSPGLKDPVARKIRSATDDS : 144 160 * 180 * 200 * 220 E. coli : WVQRFLTEP----LTAVFADWYQQPVFASLNDDQRRELVALR---SNNNGATLAAMLEATSLAVQPDLRANLSAR : 198 Nostoc : IARKLSRSILQIDFAAFLSNWYNQPIFGYIKNHPQYDRMIEN--RLQNNPQELDKSLRFMGTGCQPSLWEKLQEN : 211 Listeria : LADTLDADG----IEPFVAYWENLALFASQKNLPFALKKRIRLERLAQNPHGLAKSLRGMGTGKQPSYWENLADF : 209 Trichodesm : LANKLE----NSNFKEFLINWYNQPLFESLRQHNNFEKLIER--RLENNPLELGKSLRNLGIGNQPSLWNKLSNH : 208 AtMenH : KARMMVDNG----LYIFIENWYNGGLWKSLRNHPHFSKIAASR-LLHGDVPSVAKLLSDLSSGRQPSLWEELEDC : 214 * 240 * 260 * 280 * 300 E. coli : TFAFYYLCGERDSKFRALAAELAADCHVIP---RAGHNAHRENPAGVIASLAQILRF--- : 252 Nostoc : KIHILLLTGEYDKKFISINTEIAQICEFAQ---LKIIKNAGHNIHFENTLAFVENIKDFLSTAN--- : 272 Listeria : TFPVLLITGNLDEKFEKIAREMKQLLPNST---HVTVQEAGHAVYLEQPNIFSSQLIYWLEGILKEEEK- : 275 Trichodesm : QIPTLLMVGKYDHKFKAINTEIAELCLPAK---LKVIGESGHNIHWENPREWMETIINFLIKGKKKTNIS : 275 AtMenH : DTNISLVFGEKDVKYKQIATRMYREMSKSKKSVNNIIEIVEIPEAGHAVHLESPLRVILALRKFLTRVHNSSTET : 289 * E. coli : --- : - Nostoc : --- : - Listeria : --- : - Trichodesm : LKNTTRVTFF--- : 285 AtMenH : ELSQKLLLALKEM : 302

Figure 17. Alignment of the Arabidopsis thaliana MenH module of PHYLLO (AtMenH) with MenH proteins of the representative eubacteria Escherichia coli K12 (E. coli, accession number AAC75323), Nostoc punctiforme (Nostoc, ZP_00112005), Listeria innocua (Listeria, CAC97013), and Trichodesmium erythraeum IMS101 (Trichodesm, ZP_00071896.1). Residues with black background (X) -100% conservation, grey background (X)- 60% conservation, grey background with black letter (X) -30% conservation. Residues coloured in red correspond to proposed catalytic triad discussed in the text and depicted in the Fig. 16.