Premium
High‐resolution structure and biochemical properties of a recombinant Proteus mirabilis catalase depleted in iron
Author(s) -
Andreoletti Pierre,
Sainz Germaine,
Jaquinod Michel,
Gag Jean,
Jouve Hélène Marie
Publication year - 2002
Publication title -
proteins: structure, function, and bioinformatics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.699
H-Index - 191
eISSN - 1097-0134
pISSN - 0887-3585
DOI - 10.1002/prot.10283
Subject(s) - heme , catalase , proteus mirabilis , chemistry , tetramer , enzyme , protein quaternary structure , stereochemistry , protoporphyrin ix , biochemistry , recombinant dna , escherichia coli , organic chemistry , protein subunit , photodynamic therapy , gene
Heme catalases are homotetrameric enzymes with a highly conserved complex quaternary structure, and their functional role is still not well understood. Proteus mirabilis catalase (PMC), a heme enzyme belonging to the family of NADPH‐binding catalases, was efficiently overexpressed in E. coli . The recombinant catalase (rec PMC) was deficient in heme with one‐third heme and two‐thirds protoporphyrin IX as determined by mass spectrometry and chemical methods. This ratio was influenced by the expression conditions, but the enzyme‐specific activity calculated relative to the heme content remained unchanged. The crystal structure of rec PMC was solved to a resolution of 2.0 Å, the highest resolution obtained to date with PMC. The overall structure was quite similar to that of wild‐type PMC, and it is surprising that the absence of iron had no effect on the structure of the active site. Met 53 close to the essential His 54 was found less oxidized in rec PMC than in the wild‐type enzyme. An acetate anion was modeled in an anionic pocket, away from the heme group but important for the enzymatic reaction. An alternate conformation observed for Arg 99 could play a role in the formation of the H‐bond network connecting two symmetrical subunits of the tetramer. Proteins 2003;50:261–271. © 2002 Wiley‐Liss, Inc.