Part:BBa_K535001

HydG-> Chlamydomonas reinhardtii

Hydrogenase H-cluster assembly is necessary to have an active hydrogenase expression, because of the complexity of hydrogenases’ active sites, they require maturation machineries that involve metal-ion transport, they require at least three accessory proteins called the HydE, HydF, and HydG maturases. All organisms with [2Fe] hydrogenase and sequenced genomes contain homologues of HydE, HydF, and HydG.

Specifically HydG is a bi-functional enzyme that uses a relatively simple substrate to generate the triple-bonded ligands of [FeFe]-hydrogenase active site.

We will use the maturases (HydEF and HydG) from Chlamydomonas reinhardtii. In the C. reinhardtii genome, this genes are adjacent and are arranged in an order suggestive of divergent expression from the same promoter region.

HydEF and HydG genes are transcribed anaerobically in parallel with the HydA1 and HydA2 [Fe] hydrogenase genes in C. reinhardtii.

In prokaryotic genomes HydE, HydF, and HydG are found in putative operons with [Fe] hydrogenase structural genes. HydG belongs to the emerging radical S-adenosylmethionine (commonly designated “Radical SAM”and also known as the AdoMet radical) superfamily of proteins. These proteins participate in numerous biochemical reactions including but not limited to sulfur insertion, radical formation, organic ring synthesis, and anaerobic oxidation.

HydG contain the signature Cys-X3-Cys-X2-Cys motif that is typically found within the Radical SAM protein superfamily. This motif coordinates a redox active [4Fe4S] cluster under reducing conditions.

HydG also uses tyrosine to synthesize the CN-/CO ligands of [FeFe]-hydrogenase active site, initial steps of CN- synthesis are carried out in the TIM-barrel domain of HydG but some component(s) of the CTD (C-terminal domain) are later needed. Mutation of any of the three conserved cysteines found in the CTD of HydG resulted in an inactive hydrogenase, which indicates that CO synthesis is metal-based and occurs in the CTD and probably uses the [4Fe–4S] cluster.

Sequence and Features

Assembly Compatibility:

• 10
  COMPATIBLE WITH RFC[10]
• 12
  COMPATIBLE WITH RFC[12]
• 21
  COMPATIBLE WITH RFC[21]
• 23
  COMPATIBLE WITH RFC[23]
• 25
  INCOMPATIBLE WITH RFC[25]

  Illegal NgoMIV site found at 53
  Illegal NgoMIV site found at 68
  Illegal NgoMIV site found at 164
  Illegal NgoMIV site found at 311
  Illegal NgoMIV site found at 977
  Illegal NgoMIV site found at 1085
  Illegal NgoMIV site found at 1298
  
• 1000
  INCOMPATIBLE WITH RFC[1000]

  Illegal BsaI site found at 820
  Illegal BsaI.rc site found at 218
  Illegal BsaI.rc site found at 284
  Illegal BsaI.rc site found at 881